Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo - PubMed (original) (raw)
Urokinase receptor and fibronectin regulate the ERK(MAPK) to p38(MAPK) activity ratios that determine carcinoma cell proliferation or dormancy in vivo
J A Aguirre-Ghiso et al. Mol Biol Cell. 2001 Apr.
Free PMC article
Abstract
We discovered that a shift between the state of tumorigenicity and dormancy in human carcinoma (HEp3) is attained through regulation of the balance between two classical mitogen-activated protein kinase (MAPK)-signaling pathways, the mitogenic extracellular regulated kinase (ERK) and the apoptotic/growth suppressive stress-activated protein kinase 2 (p38(MAPK)), and that urokinase plasminogen activator receptor (uPAR) is an important regulator of these events. This is a novel function for uPAR whereby, when expressed at high level, it enters into frequent, activating interactions with the alpha5beta1-integrin, which facilitates the formation of insoluble fibronectin (FN) fibrils. Activation of alpha5beta1-integrin by uPAR generates persistently high level of active ERK necessary for tumor growth in vivo. Our results show that ERK activation is generated through a convergence of two pathways: a positive signal through uPAR-activated alpha5beta1, which activates ERK, and a signal generated by the presence of FN fibrils that suppresses p38 activity. When fibrils are removed or their assembly is blocked, p38 activity increases. Low uPAR derivatives of HEp3 cells, which are growth arrested (dormant) in vivo, have a high p38/ERK activity ratio, but in spite of a similar level of alpha5beta1-integrin, they do not assemble FN fibrils. However, when p38 activity is inhibited by pharmacological (SB203580) or genetic (dominant negative-p38) approaches, their ERK becomes activated, uPAR is overexpressed, alpha5beta1-integrins are activated, and dormancy is interrupted. Restoration of these properties in dormant cells can be mimicked by a direct re-expression of uPAR through transfection with a uPAR-coding plasmid. We conclude that overexpression of uPAR and its interaction with the integrin are responsible for generating two feedback loops; one increases the ERK activity that feeds back by increasing the expression of uPAR. The second loop, through the presence of FN fibrils, suppresses p38 activity, further increasing ERK activity. Together these results indicate that uPAR and its interaction with the integrin should be considered important targets for induction of tumor dormancy.
Figures
Figure 1
Production and extracellular organization of FN in tumorigenic and dormant HEp3 cells. (A**)** Total cellular FN of cells grown in FN-depleted FBS for 6 h (left) and 48 h (middle) and lysed or secreted FN (right) were determined by SDS-PAGE under reducing conditions and Western blot. The arrow indicates monomeric FN. LK25 and T-HEp3 tumorigenic cells; AS24 and D-HEp3 dormant cells (see MATERIALS AND METHODS). (B**)** Western blot of insoluble FN matrix. Deoxycholate-soluble and -insoluble fractions (50 μg of protein) of T-HEp3 and D-HEp3 cells were separated by SDS-PAGE under reducing or nonreducing conditions and tested for FN by Western blotting. β-ME, β-mercaptoethanol. The arrows indicate dimeric or monomeric FN. (C**)** Confocal laser scanning IF (XY sections) for FN. Tumorigenic (T-HEp3/LK25) cells or dormant (D-HEp3/AS24) cells were plated in FN-depleted FBS supplemented with 5 μg/ml human FN and, after 16 h**,** fixed and stained with anti-human FN antibody. The arrows indicate FN fibrils that appeared to be mostly apical but also basolateral and in many cases formed cell-cell bridges. Staining for FN and visualization of nuclei was also performed on histological sections of T-HEp3 or D-HEp3 cells maintained in vivo on the CAM for a week (right). Note the absence of FN matrix deposition in dormant cell lines in culture and in vivo. (D) Quantitation of FN fibril-positive cells. Cells were double-stained with anti-FN antibodies (as in C) and with DAPI. The number of FN fibril-positive cells is expressed as a percentage of DAPI-stained cells. (E) Colocalization of FN and F-actin by confocal IF microscopy. LK25 cells were grown as in C and, after fixing, triple-staining for FN (green), F-actin (red), and nuclei (blue) was performed. The yellow signal indicates colocalization of FN and F-actin. Note that the fibrillar or globular FN colocalizes with the F-actin cytoskeleton and that FN fibrils extend beyond the cell body (see also Figure 2A). The images in C and E are representative of the most prevalent cell types. Bars, 40 μm.
Figure 2
The effect of disruption of uPAR-integrin association on FN fibrillogenesis. (A) LK25 cells plated on gelatin (10 μg/ml)-coated coverslips for 60 min were preincubated with medium alone (a) or with antibodies to α5β1-integrins (BIIG2 20 μg/ml, b), β1-integrin (AIIB2 10 μg/ml, c), uPAR (epitope in domain-III, R2, 10 μg/ml, d), CD55 (CD55/DAF, 15 μg/ml, e), or medium with 50 μM peptide p25 (f), for 20 min (AIIB2 and BIIG2 are function-blocking antibodies). Human FN (5 μg/ml) was then added to all wells, and the cells were incubated overnight to allow for FN fibrillogenesis. Cells were examined by IF confocal laser scanning microscopy. The arrows indicate FN fibrils between or beyond cell bodies. (B) Quantitation of FN fibril-positive cells. At least 200 cells were scored per treatment in duplicate. Treatments in the left graph are: C, control medium; CD55, antibody anti-CD55 (10 μg/ml); R2, uPAR antibody (10 and 20 μg/ml); BIIG2, anti-α5β1 antibody (20 μg/ml); AIIB2, anti-β1 antibody (10 μg/ml). Treatments in right graph are: P25, peptide 25 that disrupts uPAR-integrin interaction (5, 50, and 100 μM). Quantitation of FN fibril-positive cells was performed as described in Figure 1, C and D, using DAPI staining. Bar, 40 μm
Figure 3
Disruption of FN fibrillogenesis alters the p38/ERK activity ratio. (A) Basal levels of active and total p38 in LK25 and AS24 cells, serum-starved for 24 h (left) or basal levels of active and total ERK in T-HEp3 and D-HEp3 cells serum-starved for 24 h (right) were detected by Western blotting using anti-phospho-p38 (P-p38) and p38 antibodies (p38) or anti-phospho-ERK (P-ERK1/2) and anti-ERK (ERK2) antibodies. (B) Active p38 in T-HEp3 (lane 1), D-HEp3 (lane2), LK25 (lane3), or AS24 (lane 4) cells was detected by IP with anti-phospho-p38 antibodies and blotting with anti-p38 antibodies (top, P-p38). Middle, light chain of IgG (Ig-LC), as a gel-loading control. Bottom (p38), total p38 in cell lysates used for IP as detected by Western blotting. The numbers below each lane show the P-p38 to p38 ratio. (C) Effect of blocking FN fibrillogenesis or disruption of uPAR-integrin complex on p38 activation. Cells were plated in gelatin-coated dishes and treated with anti-β1 or anti-α5β1 antibodies as indicated in Figure 2A (T-HEp3, left) or with anti-β1 and anti-uPAR antibodies (LK25, right). The cells were lysed and active p38 was detected as in B. c, control (no antibody); cells treated with antibodies to: β1 (AIIB2, 10 μg/ml), α5β1 (BIIG2, 20 μg/ml), or uPAR (R2, 10 μg/ml). Control LK25 cells always displayed a slightly higher level of active p38 than T-HEp3 cells (see B and C). (D) Effect of FN fibrils disruption with FN III1-C fragment on p38 activation. LK25 cells were plated in FN-depleted FBS supplemented with 5 μg/ml human FN. After overnight incubation to allow for FN fibrillogenesis, the cells were left untreated (control, a and d) or were treated for 16 h with 20 μM III1-C (b and e) or III11-C (c and f) fragments of FN. Cells were then fixed and stained for either FN (a–c) or with anti-p38 monoclonal antibodies (d–f) and visualized by standard IF. The arrows indicate FN fibrils present only in untreated or control (III11-C) -treated cells (a and c) or p38 in the nucleus (e) after FN fibrils disruption with III1-C FN fragment. Bars, 40 μm.
Figure 4
Analysis of cross-talk between ERK and p38 pathways. (A) Effect of p38 inhibition on ERK activation (short-term treatment). Serum-starved T-HEp3 (left) or D-HEp3 (right) cells were treated for 5 or 20 min with 0, 2, and 5 μM SB203580 in DMSO, then lysed, and assayed by Western blotting using anti-phospho-ERK for active ERK or anti-ERK antibodies for total ERK. (B) Long-term treatment. D-HEp3 cells were treated in the absence of serum for 5, 24, and 48 h with 2 μM SB203580 (+) or DMSO (−) and assayed after lysis for ERK activation as indicated in A. After 48 h of treatment some cultures were washed free of the inhibitor and incubated for an additional 24 and 72 h in serum-free medium (lanes indicated as wash-out) before lysing and testing for active ERK. (C) AS24 cells were treated as in B: DMSO treated (−) for 5 or 24 h or SB203580 treated (+) for 5, 24, and 48 h; some plates were treated with the inhibitor for 48 h, washed, and incubated in serum-free medium for an additional 24 h (wash out). (D) Effect of the stable expression of a dominant negative p38 on ERK activation. Pools of D-HEp3-neo or D-HEp3-p38DN cells were lysed and the levels of active and total ERK, as well as the levels of FLAG protein, were detected by Western blotting as in A and compared with the level of active ERK in parental, tumorigenic T-HEp3 cells.
Figure 5
Effect of p38 inhibition on uPAR and α5β1 expression (A) uPAR-mRNA level. RNA extracted from D-HEp3 cells treated with DMSO (−) or 2 μM SB203580 in DMSO (+) for 5, 24, and 48 h were tested by Northern blot using 32P-labeled uPAR-cDNA as a probe. Bottom, 36B4 mRNA used as a loading control. The graph shows the ratio of uPAR/36B4 signals, measured by laser scanning densitometry. (B) uPAR-protein level. D-HEp3 cells were incubated with 2 μM SB203580 in DMSO (+) or DMSO (−) for 5, 16, 24, or 48 h and, after lysis, the cells were processed and analyzed by Western blotting for uPAR. Total ERK served as a loading control. (C) uPAR protein levels in cells expressing a dominant negative p38. D-HEp3-neo or D-HEp3-p38DN cells were lysed and the levels of uPAR, ERK (as loading control), or p38DN were detected by Western blot. (D) Effect of Mek inhibition on SB203580-induced ERK activation and uPAR up-regulation. D-HEp3cells untreated, or treated with 2 μM SB203580 with or without 30 μM PD98059 (Mek inhibitor) for 36 h, were lysed, and the levels of uPAR protein (top) as well as active (middle) and total (bottom) ERK levels were detected by Western blot. The levels of uPAR and active ERK in T-HEp3 cells served as positive controls. (E) FACS analysis for α5β1-integrin surface expression using anti-α5β1-integrin antibodies (clone HA5) in T-HEp3, D-HEp3, or AS24 cells treated with the p38 inhibitor SB203580 (2 μM) for 48 h (SB-D-HEp3 or SB-AS24) or untreated (C-T-HEp3, C-D-HEp3, C-AS24). Isotype-matched IgG was used as control. The numbers indicate the percentages of cells positive for surface α5β1-integrin. (F) Coimmunoprecipitation of uPAR and α5β1-integrin in SB203580-treated cells. D-HEp3 cells untreated or treated with 2 μM SB203580 or 2 μM SB203580 and 30 μM PD98059 were lysed, and the cell lysates were subjected to IP with anti-α5β1-integrin antibodies and the immunoprecipitated proteins were tested by Western blotting with antibodies to β1-integrin and uPAR.
Figure 6
Effect of p38 inhibition on uPAR and β1-integrin surface expression. Untreated T-HEp3 cells (A–C) or D-HEp3 cells treated for 48 h with 2 μM SB203580 (G–I) or DMSO alone (D–F) were fixed and stained, without permeabilization, for β1-integrin (A, D, and G, AIIB2 antibody, green) or uPAR (B, E, H, R2 antibody, red), as described in MATERIALS AND METHODS, and detected by confocal laser scanning IF microscopy. C, F, and I, show the merged image of the uPAR and β1-integrin signals; yellow indicates colocalization of uPAR and β1-integrin signals. A to I are XY sections; insets in C, F, and I are XZ sections of the same cells showing the overlay. Note the high surface (apical and basolateral) expression of uPAR in D-HEp3 cells treated with the p38 inhibitor (compare E with H) that colocalizes with β1-integrin signal (compare F with I and the corresponding insets), which remains unchanged in SB203580-treated cells (compare D and G). Bars, 40 μm.
Figure 7
Analysis of the effect of p38 inhibition on α5β1-integrin function. (A) Adhesion of DMSO- or SB203580-treated cells to FN. D-HEp3 cells were treated for 48 h with 0 or 2 μM SB203580 in DMSO, detached, and plated onto FN-coated (0.5–5 μg/ml) wells. After 10 min, the cells were fixed and stained and the attached cells were quantitated as described in MATERIALS AND METHODS. (B) a to c, confocal IF microscopy (a–c, XY sections) for FN fibrils in D-HEp3 cells treated with DMSO alone (a), 2 μM SB203580 (b), or SB203580 with 5 μg/ml human FN (c). Bar, 40 μm. d to f, effect on FN fibril formation of transient transfection of uPAR in D-HEp3. Cells transfected with empty vector (d) or with the vector encoding uPAR (e and f) and grown in serum-containing medium were fixed and stained for FN. Note that only cells transfected with uPAR are able to organize FN into thick bundles and fibrils. A larger magnification of the fibrils is shown in f. Bar, 20 μm. The arrows indicate fibrils. (C) Quantitation of the effect of p38 inhibition on FN fibrillogenesis. Cells (D-HEp3 or AS24) untreated (control) or treated with SB203580 (SB 2 μM) were incubated for 48 h with or without 5 μg/ml FN (FN and SB 2 μM + FN). Cells positive for FN fibrils are shown as percentages of cells with DAPI-positive nuclei.
Figure 8
Relevance of uPAR/integrin signaling to ERK and p38 activity in vivo. (A) Down-modulation of ERK activity in vivo by disruption of the uPAR-β1-integrin interaction. T-HEp3 or LK25 cells were transiently transfected with an HA-ERK2 construct (lanes 2, 3, 5, and 6) or mock-transfected (lanes 1 and 4) for 24 h, detached, and incubated in suspension for 30 min with 10 μg/ml R2 and AIIB2 antibodies (lanes 3 and 6) or without antibodies (lanes 2 and 5) and inoculated (2.5 × 106 cells/CAM) onto chick embryo CAMs for an additional 24 h. After in vivo growth, the tumor cell-containing CAM tissue was excised and lysed (see MATERIALS AND METHODS). The lysates were immunoprecipitated with anti-HA antibodies. Total HA-ERK expression (IP: HA, WB: HA) or phosphorylated HA-ERK (IP: HA, WB: P-ERK) was detected by Western blot. (B) Transient expression of uPAR in spontaneous dormant D-HEp3 cells restores high ERK activation. D-HEp3cells were transiently transfected with 2 μg of uPAR-cDNA or control plasmids and uPAR expression or ERK activation were examined by Western blot of whole cell lysates (top three panels). In the bottom two panels D-HEp3 cells were transiently cotransfected with empty vector or HA-ERK2 (1 μg) constructs or with increasing amounts (0.5–2 μg) of uPAR cDNA plasmid. HA-ERK was immunoprecipitated with anti-HA and blotted with anti-phospho-ERK or with anti-HA antibodies. Note that uPAR expression generates a strong activation of ERK. (C) Effect of transient expression of uPAR on the growth of D-HEp3 cells in vivo. D-HEp3 cells transfected with empty vector or with the uPAR-encoding vector were grown for 48 h and after detachment the cells were inoculated onto 9-d-old CAMs. The number of cells per tumor was determined as described in MATERIALS AND METHODS. The horizontal lines indicate the mean. *P < 0.02 as determined by Mann-Whitney test. (D) Effect of p38 inhibition on in vivo growth of dormant cells. D-HEp3 cells pretreated with 2 μM SB203580 or left untreated for 48 h or D-HEp3-neo and D-HEp3-p38DN cells were detached and inoculated at 4 × 105 cells per 9-d-old chick embryo CAM. After 3, 4, and 7 d the number of tumor cells in the tumor nodules was quantitated as described in MATERIALS AND METHODS. The graph shows the mean number and SE of the population cell divisions. Note that only D-HEp3 cells treated with SB203580 or D-HEp3-p38DN cells developed growing tumors. Their growth rate was similar to that of T-HEp3 cells, which divided ∼3.2 times in 7 d and were not affected by SB203580 treatment (approximately three divisions); *P < 0.001 Mann-Whitney test. Unlike D-HEp3 cells, AS24 cells were unable to form growing tumors even after treatment with 2–4 μM SB203580 for 48 h. (E) Effect of anti-uPAR antibodies on the growth of D-HEp3 cells in which p38 activity was inhibited. D-HEp3-p38DN or D-HEp3 cells pretreated with 2 μM SB203580 for 48 h were incubated in suspension with 10 μg/ml anti-uPAR antibody (R2) or left untreated. The cells were then inoculated onto 9- to 10-d-old chick embryo CAMs and after 4 d the number of cell per tumor nodules was determined. These groups were compared with the growth of parental, untreated D-HEp3 or D-HEp3-neo cells. The results are expressed as percentages of control (mean and SD). *P < 0.01, D-HEp3- p38DN or D-HEp3-SB2 μM versus D-HEp3-neo or -control cells. #P < 0.01, R2 antibody treated versus untreated D-HEp3-p38DN or D-HEp3-SB2 μM cells as determined by Mann-Whitney test.
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