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Cell Growth & Differentiation Vol. 13, 141-148, March 2002
© 2002 American Association for Cancer Research

Role of the Src Homology 2 Domain-containing Protein Shb in Murine Brain Endothelial Cell Proliferation and Differentiation1

Lingge Lu, Kristina Holmqvist, Michael Cross and Michael Welsh2

Departments of Medical Cell Biology [L. L., K. H., M. W.], and of Genetics and Pathology, Rudbeck Laboratory [M. C.], Uppsala University, 751 23, Uppsala, Sweden


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To study the role of the Src homology 2 (SH2) domain-containing protein Shb in angiogenesis, wild-type Shb and SH2 domain-mutated Shb (R522K Shb) were overexpressed in murine immortalized brain endothelial cells. The wild-type Shb cells exhibited an increased rate of apoptosis on serum withdrawal. Both wild-type Shb and R522K Shb cells exhibited enhanced spreading concomitant with cytoskeletal rearrangements that occurred independently of fibroblast growth factor (FGF)-2 stimulation. However, these effects may partly be caused by altered regulation of Rac1 and Rap1 activation in the Shb cells. The Shb-induced cytoskeletal rearrangements were not dependent on phosphatidylinositol 3' kinase activity, but could be reversed by inhibition of Src family kinases. FGF-2 failed to further enhance migration of wild-type Shb and R522K Shb cells. The R522K Shb cells cultured in collagen gels exhibit diminished tubular morphogenesis when treated with FGF-2, implicating the need for a functional Shb molecule in this process. These data suggest that Shb plays a role in the proliferation and differentiation of endothelial cells and, hence, participates in angiogenesis.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Angiogenesis is the process in which new blood capillaries are formed from preexisting vessels, and it occurs in both physiological and pathological situations such as reproduction, wound healing, rheumatoid arthritis, diabetic retinopathy, and tumor development (1) . Growth factors such as FGF-23 and VEGF are major regulators of angiogenesis, which involves proliferation, migration and differentiation of endothelial cells (2 , 3) .

Shb is an adaptor protein with proline-rich motifs in its NH2 terminus, a central phospho-tyrosine binding domain, and a COOH-terminal SH2 domain (4) . It has previously been shown that Shb interacts with the PDGF ß-receptor, FGF receptor-1, and T-cell receptor via its SH2 domain and participates in tyrosine kinase-dependent signaling through the formation of multiprotein complexes (5 , 6) . Overexpression of Shb induces increased apoptosis on serum withdrawal in NIH3T3 cells (7) and growth factor-stimulated differentiation in PC12 cells (8) . Stimulation with FGF-2 increases Shb tyrosine phosphorylation in bovine adrenal cortex capillary endothelial cells (9) and murine IBE cells (10) . To further address the role of Shb in angiogenesis, wild-type Shb and SH2 domain-mutated Shb (R522K Shb) were overexpressed in IBE cells. We describe a role for Shb in IBE cell proliferation and differentiation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Proliferation and Apoptosis of IBE Cells Overexpressing Shb.
Overexpression of Shb in the IBE cell clones was confirmed by Western blot analysis (Fig. 1A)Citation . The clones displayed a 2- to 4-fold increase in the Shb content compared with the vector control clones. Probing the same blot for Shc, Grb2, and ERK revealed no differences in the content of these proteins. When the cells were maintained in 15% serum at 33°C, no significant differences of the proliferation rates were observed among the wild-type Shb, R522K Shb, and the control IBE cells (Fig. 1B)Citation , although the wild-type Shb cells tended to proliferate at a lower rate than the control cells. The data in Fig. 1BCitation are based on the results obtained from using two independent clones in each group. The wild-type Shb cells displayed a significantly lower rate of DNA synthesis compared with the control cells in the presence of serum (Fig. 1C)Citation . Shb has previously been shown to induce apoptosis in NIH3T3 cells and RINm5F cells when maintained in low serum (7 , 11) . We thus, examined the rate of apoptosis in Shb-overexpressing IBE cells after 3 days of serum deprivation by use of flow cytometry after labeling the cells with Annexin-V. As shown in Fig. 1DCitation , wild-type Shb IBE cells displayed a rate of 34.0 ± 5.5% apoptotic cells, which was significantly higher than the corresponding values in R522K Shb cells (13.4 ± 2.4%) and the control IBE cells (18.6 ± 3.0%). Thus, Shb overexpression also causes elevated apoptosis in IBE cells when they are deprived of serum. No differences in the rate of apoptosis between the groups were observed when cultured in the presence of 15% serum (data not shown).



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Fig. 1. Expression levels, proliferation, DNA synthesis, and apoptosis of IBE cells overexpressing Shb. A, overexpression of wild-type and R522K Shb in IBE cells. IBE cells transfected with wild-type or R522K Shb or with empty vector were subjected to Western blot analysis. The blots were probed with anti-Shb antibody, stripped, and reprobed with anti-Grb2, anti-Shc, and anti-ERK. B, IBE cells overexpressing wild-type Shb or R522K Shb were cultured in Ham’s F-12, 15% FBS, and 20 units/ml IFN-{gamma} at 33°C. Cell numbers were scored on 5 consecutive days. Means ± SE are given for two observations and are based on two separate clones in each group. C, IBE cells overexpressing both wild-type Shb and R522K Shb were cultured in 15% serum. After 18 h, the cells were labeled with 1 µCi/ml [3H]thymidine for 4 h. Incorporated [3H]thymidine was quantified by scintillation counting. The DNA contents of the samples were measured. *, P < 0.05 when compared with control. Means ± SE for six to nine observations are given and are based on three separate clones in each group. D, apoptosis in Shb overexpressing IBE cells. IBE cells overexpressing both wild-type Shb and R522K Shb were starved in serum-free medium, labeled with Annexin-V, and subsequently analyzed by flow cytometry. *, P < 0.05 compared with the control. Means ± SE for nine observation are given for three different clones in each group. kDa, Mr in thousands.

 
Activation of PKB/Akt.
The serine/threonine protein kinase PKB/Akt is known to transmit survival signals, and activation of this kinase is paralleled by its phosphorylation (12 , 13) . To address its putative role for the apoptotic response of the wild-type Shb IBE cells, the different cells were stimulated with FGF-2 for various times, and the activation of PKB/Akt was then examined. FGF-2 induced activation of PKB/Akt to a similar degree in all of the cells at all of the time points (Fig. 2)Citation . The basal level of PKB/Akt phosphorylation also appeared to be similar in the different clones, which suggests that altered PKB/Akt activity is not responsible for the increased apoptosis of the Shb IBE cells.



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Fig. 2. Activation of PKB/Akt. A, IBE cells were starved as above. After being stimulated with 10 ng/ml FGF-2 for the indicated times, the cells were lysed in SDS sample buffer and subjected to Western blot analysis. The blots were probed with anti-phospho-Akt or anti-total-Akt antibodies. B. relative activation of Akt was quantified by relating densitometric values of phospho-Akt with those of total-Akt. The means ± SE from two to four independent experiments are given.

 
Overexpression of Shb Induces Cell Spreading on Gelatin.
Overexpression of wild-type Shb in IBE cells seeded on gelatin lead to an increased cell spreading compared with control cells (Fig. 3)Citation . IBE cells expressing R522K Shb also showed an increased spreading compared with control cells, although not to the same extent as the wild-type Shb cells.



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Fig. 3. Overexpression of Shb induces cell spreading on gelatin. IBE cells were seeded and maintained in Ham’s F-12 supplemented with 15% FBS on gelatin-coated dishes for 2 h. The photographs were taken using a Nikon TMS microscope. The data are representative for three different clones in each group.

 
IBE Cells Overexpressing Shb Exhibit Cytoskeletal Rearrangements.
Growth factors such as PDGF, insulin, and EGF have been shown to induce lamellipodia or membrane ruffles in Swiss 3T3 fibroblasts (14) . FGF-2 stimulation leads to a slight increase in stress fiber formation rather than formation of lamellipodia in the control IBE cells (Fig. 4)Citation . The wild-type Shb cells exhibited irregular, densely stained patches, which were increased when treated with FGF-2. The R522K Shb cells displayed the same structure but to a lesser extent. These patches were similar to the giant ruffles (irregular multilayered actin structure) that appeared on the dorsal side of PDGF-stimulated PAE cells expressing Y778F mutant PDGF ß-receptor (15) . PDGF induces Rac1 activation and ruffle formation, and this process is inhibited by the PI-3 kinase inhibitor LY294002 (16 , 17) . To assess a role of PI-3 kinase-dependent Rac1 activation in the Shb-dependent changes, cells were pretreated by LY294002 before FGF-2 addition. In the control cells, FGF-2 failed to cause stress fiber formation. However, the patchy staining pattern of the wild-type Shb cells remained, which suggests that this is independent of PI-3 kinase activity. The R522K Shb cells displayed a somewhat reduced content of stress fibers. Alternatively, the cytoskeletal changes could reflect Src activation (18) . To address this possibility, cells were treated with the Src-family kinase inhibitor PP2 (19 , 20) . PP2 reverted all of the FGF-2 induced changes in the control cells but also prevented the Shb-dependent patchy staining pattern in the wild-type Shb cells. Thus, FGF-2 causes both PI-3 kinase/Rac1 and Src-dependent cytoskeletal changes in control IBE cells, whereas Shb induces an altered staining pattern that is PI-3-kinase-independent but requires Src activation.



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Fig. 4. IBE cells overexpressing Shb exhibit FGF-2 independent cytoskeletal rearrangement. IBE cells on fibronectin-coated culture slides were kept in Ham’s F-12 containing 0.1% BSA for 4 h and treated with or without 100 ng/ml FGF-2 for 5 min. To inhibit PI-3 kinase or Src-family kinase, cells were pretreated with 30 µM LY204002 or 2 µM PP2 for 30 min before the addition of FGF-2. The cells were fixed in paraformaldehyde and stained with rhodamine phalloidin. The slides were examined using a fluorescence microscope.

 
FGF-2 Failed to Promote Chemotaxis in Shb-overexpressing IBE Cells.
To assess the role of Shb on chemotaxis, IBE cells overexpressing wild-type Shb and R522K Shb, together with control IBE cells, were examined using Boyden chamber analysis in the presence or absence of FGF-2. As shown in Fig. 5Citation , FGF-2 could induce distinct migration of control IBE cells as observed previously (21) . FGF-2 stimulation failed to further increase the migration in both wild-type Shb and R522KShb cells, mainly because of elevated basal cell migration.



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Fig. 5. FGF-2 fails to promote chemotaxis in Shb overexpressing IBE cells. The IBE cells in Ham’s F-12 containing 0.25% BSA were seeded on collagen-coated filter in upper (Boyden) chambers. The lower chambers were filled with Ham’s F-12 containing 0.25% BSA with or without 10 ng/ml FGF-2. After incubation at 33°C for 4 h, the filters were fixed in ethanol, washed, and stained with Giemsa solution. The cells on the upper surface of the filters were wiped away, and the number of the cells on the lower surface of the filters were counted under a microscope. The number of untreated control cells was set to 100%. Data are shown as mean ± SE for five independent experiments. *, P < 0.05.

 
IBE Cells Overexpressing Wild-Type Shb Form Tubular Structures in Response to FGF-2 in the Tube Formation Assay.
Shb, with a functional SH2 domain, is required for proper tubular morphogenesis, because IBE cells expressing R522K Shb, form disorganized tubes. Of the four clones repeatedly analyzed, only one gave structures reminiscent of tubes but to a lesser degree than in the control cells (Fig. 6A)Citation . These tubular structures were less regular and frequent than those of the control cells. The formation of tubular structures was prominent in wild-type Shb IBE cells (Fig. 6A)Citation and could be detected already after 4 h (Fig. 6B)Citation .



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Fig. 6. Functional Shb is required for proper FGF-2-induced tubular morphogenesis. IBE cells were seeded in a collagen matrix for 48 h (A) or 4 h (B) in the absence (untreated) or presence of 5 ng/ml FGF-2. Tube formation was analyzed by a Nikon Eclipse microscope and photographed using a digital camera. The figure is representative for three different clones in each group.

 
Rac1 and Rap1 Are Differentially Regulated in Shb-overexpressing IBE Cells.
The Rac1-small GTPase functions in multiple cellular processes, including lamellipodia formation and membrane ruffling, gene transcription, cell cycle progression, and cell adhesion (22 , 23) . We assessed the activation of Rac1 in Shb-overexpressing IBE cells. In three independent experiments, FGF-2 stimulation for 2 min slightly activated Rac1 in control IBE cells but not in wild-type Shb cells. FGF-2 induced Rac1 activation in R522K Shb cells to a degree similar to that in the control cells (Fig. 7A)Citation . This transient activation was not observed when the cells were treated with FGF-2 for 5 min (results not shown). Similar results have previously been shown in PDGF-stimulated PhB fibroblasts overexpressing wild-type Shb (24) . We have described the role of Shb in Rap1 signaling in PC12 cells. Nerve growth factor-stimulated-Rap1 activation was observed only in PC12 cells overexpressing wild-type Shb (25) . In the present study, on FGF-2 stimulation for 5 min, Rap1 was activated in both wild-type Shb and R522K Shb IBE cells partly because of decreased basal Rap1 activity, but not in control IBE cells (Fig. 7B)Citation . These data suggest that Shb may also function in Rap1 signaling in IBE cells.



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Fig. 7. Activation of Rac1 and Rap1 in IBE cells. IBE cells in 10-cm dishes were treated with or without 100 ng/ml FGF-2. In A, for Rac1 assay, the cells were lysed and incubated with the GST-PAK-CD fusion protein. In B, for Rap1 assay, the cell lysates were incubated with the RalGDS-RBD fusion protein. The bound active Rac1GTP or Rap1GTP was determined by Western blot analysis. Results represent three independent experiments. Units are relative densitometric absorbance when related to lysates. The means ± SE are given.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To obtain an understanding of the role of the adaptor protein Shb for angiogenesis, IBE cells were transfected with wild-type and R522K Shb. The wild-type Shb cells exhibited a higher apoptotic rate under serum deprivation as previously observed in Shb-overexpressing NIH3T3 cells and RINm5F cells (7 , 11) . This occurred in parallel with a decrease in the percentage of cells in the S phase of the cell cycle (results not shown), suggesting that the increase in apoptosis might result from a G1 block of the cell cycle. PKB/Akt is a potential regulator of cell survival and cell cycle progression (26) . Stimulation with FGF-2 induces rapid and transient activation of PKB/Akt (12 , 13) . However, stimulation of PKB/Akt was similar in all groups of cells when treated with FGF-2. In addition, activation of ERK, another putative regulator of apoptosis (27, 28, 29) , was not affected by Shb overexpression.4

Alternatively, IBE cell cycle progression could involve the small GTPase Rac1, which regulates G1 progression through activation of cyclin D1 (30 , 31) . We observed that overexpression of wild-type Shb inhibited growth factor-induced Rac1 activation in IBE cells and PhB fibroblasts consistent with the decreased rates of DNA synthesis of the wild-type Shb cells. Rac1 is activated in a PI-3 kinase-dependent manner by PDGF (16 , 32 , 33) . Such an activation could be achieved by direct signaling either from the receptor tyrosine kinase to PI-3 kinase (34) or via Ras to PI-3 kinase (35) . However, FGF-2 stimulation only induces lower activation of PI-3 kinase, and this is thought to occur mainly via Ras (36 , 37) . Eps8 mediates the transfer of signals between Ras/PI-3 kinase and Rac by forming a complex with E3b1 and Sos-1 (38) . It is possible that overexpression of wild-type Shb disrupts Rac1 signaling, in part through down-regulation of Eps8, because Eps8 binds Shb via its Src homology 3 domain (5) and Shb may influence the expression level of Eps8 (39) . Shb could also affect Rac1 signaling by regulating Rac1-guanine nucleotide exchange factor or Rac1-GTPase-activating protein. Besides mediating effects on the cell cycle, the down-regulated Rac1 signaling may have consequences for the IBE cell cytoskeleton. Shb overexpression caused a dramatically altered cytoskeletal staining pattern. This was not PI-3 kinase/Rac1 dependent but was reverted by addition of the Src-family inhibitor PP2. Thus, it seems that Shb overexpression augments the Src signaling that causes the cytoskeletal changes observed. Indeed, Shb has previously been found to associate with Src (5) .

Overexpression of Shb in IBE cells also induces FGF-2-stimulated Rap1 activation independently of the Shb SH2 domain, unlike in nerve growth factor-stimulated PC12 cells in which the effect is Shb SH2-domain dependent (25) . This suggests that Shb may use different mechanisms to regulate Rap1 activation in different cell types. Rap1 has been shown to become activated during cell adhesion, and the Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion (40 , 41) . This is further supported by the fact that Rap1 is involved in integrin-mediated cell adhesion (42, 43, 44) . Thus, Rap1 activation could be a significant component in the increased spreading in the Shb overexpressing IBE cells. The enhanced migration in the Shb-overexpressing IBE cells is independent of FGF-2 stimulation and Shb SH2 domain. It cannot be attributed only to the role of Rac1, because the Shb SH2 domain mutation did not influence FGF-2-induced Rac1 activation, although these cells showed a rate of migration similar to that of the wild-type Shb cells. Activated Rap1 also affects the cytoskeleton through binding to AF-6, which associates with the actin cytoskeletal regulator profilin (45) . Thus, Rap1 may also play a role in regulating the Shb-induced alterations of IBE cell migration. FAK is another potential regulator of cell spreading and attachment. We have, in a separate study,5 observed that Shb overexpression causes elevated levels of FAK and increased FAK activity. Indeed, activation of Shb, FAK, and Rap1 may all be linked as previously noted in the PC12 cells (25 , 46) and may, in concert, cause the alterations in the spreading and migration observed.

The wild-type Shb cells rapidly formed well-organized tubular structures, compared with the control cells, when cultured in collagen gels in the presence of FGF-2. Because the SH2 domain was required for appropriate tube formation, it seems that FGF-2-dependent tube formation does not correlate with Rac1, Rap1, and FAK activation, nor does it correlate with spreading and migration. However, the addition of a Src-family kinase inhibitor has previously been found to prevent IBE cell tube formation (47) . Thus, if Shb augments Src-family kinase signaling, this could have consequences for tubular morphogenesis. To what extent the observed Shb-dependent cytoskeletal alterations play a role in IBE cell differentiation remains to be established. It has also been shown that a Mr 125,000 protein binds to the SH2 domain of Shb in IBE cells (10) . Stimulation with FGF-2 increased the binding as well as tyrosine phosphorylation and in vitro kinase activity of this protein. This unknown protein may play a role for Shb-dependent tube formation in IBE cells.

Taken together, although Shb induces apoptosis on serum withdrawal and decreases DNA synthesis, it promotes the differentiation of IBE cells in the presence of FGF-2 and serum.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture and Retrovirus-mediated Overexpression.
IBE cells were cultured on gelatin (Sigma Chemical Co.)- coated dishes in Ham’s F-12 (Life Technology), 15% FBS (Hyclone), and 20 units/ml IFN-{gamma} (Peprotech, Inc.) at 33°C. The wild-type Shb cDNA and R522K Shb mutant (with an inactivation of the SH2 domain) cDNA were inserted into the retroviral vector pBABE Puro and transfected into Bosc 23 cells by the LipofectAMINE method. Control transfection with vector alone was also performed. After 48 h, viral supernatant was collected and virus was added to IBE cells in the presence of Polybrene (4 µg/ml). Positive clones were selected for puromycin resistance at 48 h after infection. Overexpression of wild-type Shb and R522K Shb was verified by Western blot analysis (Fig. 1A)Citation .

Cell Proliferation and DNA Synthesis Assay.
For the proliferation assay, IBE cells overexpressing wild-type Shb, R522K Shb mutant, and control IBE cells (5 x 104) were seeded in gelatin-coated 24-well plates in Ham’s F-12 supplemented with 15% FBS and 20 units/ml IFN-{gamma}. Cell numbers were scored for 4 consecutive days. For DNA synthesis assay, 1.5 x 104 cells were seeded in 24-well plates coated with 0.1% gelatin and 10 µg/ml human fibronectin. After growth in Ham’s F-12, 15% FBS, and 20 units/ml IFN-{gamma} at 33°C for 48 h, the cells were starved with Ham’s F-12 and 0.2% BSA for another 48 h. FGF-2 (5 ng/ml; Roche) was added for 18 h. The cells were labeled with 1 µCi/ml [3H]thymidine for 4 h, then washed and sonicated, followed by trichloroacetic acid precipitation before scintillation counting. The DNA contents of the samples were measured in parallel using a fluorometric assay. For all of the assays, two to five different clones from each group were simultaneously analyzed, and the results were from three independent experiments.

Detection of Apoptosis.
Cells (2 x 104) were seeded on gelatin-coated 6-cm dishes in Ham’s F-12 supplemented with 15% FBS and 20 units/ml IFN-{gamma} overnight and starved in Ham’s F-12 containing 0.1% BSA for 48 h. Apoptosis was detected by using Annexin-V-Fluos kit (Roche). Cells were gently trypsinized and resuspended in Annexin-V-Fluorescein and propidium iodide, incubated for 10 min at room temperature, and subsequently analyzed by flow cytometry with 488 nm excitation and collecting light scatter, green and red fluorescence. Apoptotic cells were defined as cells with enhanced Annexin-V fluorescence simultaneously exhibiting normal propidium iodide staining.

Determination of PKB/Akt.
Cells in 24-well plates were seeded as described above and treated with FGF-2 (10 ng/ml) for the indicated times. The cells were lysed with SDS sample buffer containing 2 mM PMSF and were sonicated. After boiling for 5 min, the samples were subjected to SDS-PAGE. For Western blot analysis, proteins were electrically transferred onto Immobilon filter (Millipore). The filters were blocked with 5% nonfat milk in PBS, 0.1% Tween 20 and incubated with either anti-phospho-Akt or anti-total-Akt antibodies (New England Biolabs). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Cell Spreading and Actin Rearrangement.
Cells (3 x 106) were seeded on gelatin-coated dishes in Ham’s F-12, supplemented with 15% FBS at 33°C for 2 h. Photographs were taken using a Nikon TMS microscope connected to a SPOT 2 digital camera (Diagnostic Instruments). For actin rearrangement, cells cultured on fibronectin-coated culture slides (Becton Dickinson) were kept in Ham’s F-12 containing 0.1% BSA for 4 h and then stimulated with or without 100 ng/ml FGF-2 for 5 min. To inhibit PI-3 kinase or Src-family kinase, cells were pretreated with 30 µM LY204002 (Sigma Chemical Co.) or 2 µM PP2 (Calbiochem) for 30 min before the addition of FGF-2. The cells were fixed in 3% paraformaldehyde and permeabilized with acetone. After blocking with 10% FBS, the cells were incubated with rhodamine phalloidin (Molecular Probes) for 1 h. The cells were rinsed four times with PBS and antifade was added (Molecular Probes). The slides were examined using a fluorescence microscope.

Chemotaxis Assay.
The assay was performed using a Boyden chamber as described previously (48) . Filters (8 µm thick, 8 µm pore; Whatman,) were coated with 100 µg/ml type I collagen (Vitrogen 100; Collagen Corporation). IBE cells were trypsinized and resuspended at 6 x 105 cells/ml in Ham’s F-12 containing 0.25% BSA. The lower chambers were filled with Ham’s F-12 containing 0.25% BSA with or without 10 ng/ml FGF-2 and were covered with the coated filter. The cell suspension was loaded in the upper chamber and incubated for 4 h at 33°C. Filters were fixed in ethanol, washed, and stained with Giemsa solution. After removing the cells on the upper surface of the filters, the number of the cells on the lower surface of the filters were counted using a microscope. The number of untreated control cells was set to 100%. All of the experiments were performed in triplicate.

Tube Formation.
Control IBE cells and IBE cells overexpressing either wild-type or R522K Shb were cultured in gelatin-coated tissue culture flasks in Ham’s F-12 supplemented with 15% heat-inactivated FBS for 24 h, before the cells were washed in PBS, and Ham’s F-12 containing 2% FBS was added for 24 h. 24-well plates were coated with a collagen solution consisting of 10 x Ham’s F-12, collagen Type 1 (Vitrogen 100; Collagen Corporation) and 0.1 M NaOH in a ratio of 1:8:1, and incubated at 37°C for 1 h, in order for the collagen to form a gel. Ham’s F-12 medium was added to prevent cracking of the gel. The medium was aspirated before seeding the cells. Cells (1.5 x 105/well) in Ham’s F-12 and 0.2% BSA were seeded in collagen gel-coated 24-well plates and incubated at 33°C for 3 h. The cell culture medium was aspirated and a top layer of collagen solution was added. The collagen was jellied for 1 h at 37°C before adding Ham’s F-12 with or without FGF-2 at a final concentration of 5 ng/ml. The cells were then incubated at 33°C for 48 h. The cells were fixed in 2.5% glutaraldehyde/PBS and stored at 4°C. Tube formation was analyzed by a Nikon Eclipse TE 300 Microscope and photographed using a SPOT 2 digital camera (Diagnostic Instruments).

Determination of Rac1 and Rap1 Activation.
Activation of Rac1 and Rap1 were determined as described previously (24 , 25) . Subconfluent cells in 10-cm dishes were maintained in Ham’s F-12 containing 0.1% BSA overnight. For the Rac1 assay, cells were stimulated with or without 100 ng/ml FGF-2 for 2 min. The cells were lysed and incubated with the GST-PAK-CD fusion protein precoupled to glutathione-Sepharose beads. For the Rap1 assay, cells were stimulated with or without 100 ng/ml FGF-2 for 5 min. The cells were lysed and incubated with the RalGDS-RBD fusion protein precoupled to glutathione-Sepharose beads. The bound active Rac1GTP or Rap1GTP was determined by Western blot analysis.


    Footnotes
 
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.

1 Supported by grants from the Juvenile Diabetes Foundation International, the Swedish Medical Research Council (31X-10822), the Swedish Diabetes Association, the Novo-Nordisk Foundation, and the Family Ernfors Fund. Back

2 To whom requests for reprints should be addressed, at Department of Medical Cell Biology, Box 571, biomedicum, 751 23, Uppsala, Sweden. Phone: 46-18-4714447; Fax: 46-18-556401; E-mail: michael.welsh{at}medcellbiol.uu.se. Back

3 The abbreviations used are: FGF, fibroblast growth factor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; VEGF, vascular EGF; SH2, Src homology 2; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; IBE, immortalized brain endothelial (cell); PI-3, phosphatidylinositol 3'; PKB, protein kinase B; FAK, focal adhesion kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Back

4 M. J. Cross, L. Lu, P. Magnusson, D. Nyqvist, K. Holmqvist, M. Welsh, and L. Claesson-Welsh. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. Submitted for publication. Back

5 K. Holmqvist, M. J. Cross, D. Riley, and M. Welsh. The Shb adaptor protein causes Src-dependent cell spreading and activation of focal adhesion kinase in immortalized brain endothelial cells. Submitted for publication. Back

Received for publication 5/29/01. Revision received 2/ 6/02. Accepted for publication 2/13/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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