| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
vß3 Integrin through Ras/Raf-1/MEK/ERK and Calcium/CaMKII Signals
Departments of Biologia e Patologia Cellulare e Molecolare (M.I., A.L.C., S.M., E.D.V., G.R.), Endocrinologia ed Oncologia Molecolare e Clinica (F.M., G.F., M.V.), and Scienze Biomorfologiche e Funzionali (G.T.), and Unit of Endocrine and General Surgery (L.A.M.), Università Federico II, 80131 Naples, Italy; and Institute of Endocrinologia ed Oncologia Sperimentale "G. Salvatore" (G.R.), Consiglio Nazionale delle Ricerche, 80131 Naples, Italy
Address all correspondence and requests for reprints to: Mario Vitale, Dipartimento di Endocrinologia ed Oncologia Molecolare e Clinica, Via S. Pansini, 5 Napoli, 80131 Italy. E-mail: mavitale{at}unina.it.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
vß3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We recently demonstrated in the same cell type that integrin binding to FN also generates a third pathway that is calcium/calcium calmodulin-dependent kinase II (Ca2+/CaMKII). The latter modulates the Ras/Raf-1/Mek/Erk pathway by binding to Raf-1 and regulating its activity (11). Although TAD-2 cells and normal thyroid cells in primary cultures display common integrin profile and FN-induced behavior (4, 12), a direct demonstration of the existence of such integrin-generated signal in normal thyroid cells in primary cultures is lacking. Moreover, because the pattern expression of adhesion molecules changes after cell transformation, it is important to determine which integrin receptor generates the signals that mediate FN-induced proliferation.
Thus, we investigated the signal pathways generated by FN-dependent integrin activation in normal human thyroid cells in primary culture. We provide evidence that FN binding to the integrin vß3 activates the Ras/Raf-1/Mek/Erk and the Ca2+/CaMKII pathways and that both are necessary to promote FN-induced proliferation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Tissue specimens were obtained at surgery from contralateral lobes of thyroid papillary carcinomas or internodular tissue of nodular goiters of subjects undergoing thyroidectomy. Informed consent for cell culture preparation was obtained from patients undergoing thyroidectomy.
Cell cultures were prepared as previously described (4). Briefly, tissues were chopped by scalpels in small pieces and digested by type IV collagenase (Sigma Chemical Co., St. Louis, MO) 1.25 mg/ml in Ham’s F-12 medium (F-12) and 0.5% BSA overnight at 4 C under rotation. Cells were pelleted by centrifugation at 150 x g for 5 min, washed twice in BSA-F-12, seeded in petri dishes, and cultured in 5% CO2 atmosphere at 37 C in F-12 supplemented with 10% fetal calf serum (FCS). Medium was changed every 3–4 d, and the cells were harvested by treatment with 0.5 mM EDTA in calcium- and magnesium-free PBS containing 0.05% trypsin. The follicular origin of the cultures was confirmed by flow cytofluorometry searching for cytokeratin and thyroglobulin as previously described (12). More then 98% of the cells in culture were positive for cytokeratin and thyroglobulin. For each experiment, single individual cultures were used and results pooled for statistical analysis.
The TAD-2 cell line, obtained by Simian virus 40 infection of human fetal thyroid cells was generously donated by Dr. T. F. Davies (Mount Sinai, New York, NY) (13). Thyroid papillary carcinoma cell lines NPA and TPC-1 were kindly donated by M. Nagao (Tokyo, Japan) (14). All cell lines were cultured in F-12 supplemented with 10% FCS.
Coated plates were prepared as follows: the plates were filled with PBS, 1% heat-denatured BSA (Sigma, St. Louis, MO), or 100 µg/ml human FN (Collaborative Research, Bedford, MA). After overnight incubation at 4 C, the plates were washed with PBS three times and used.
Antibodies and flow cytometric analysis
For intracellular immunofluorescence (cytokeratin and thyroglobulin), cells were fixed in 3.5% paraformaldehyde, 0.2% Tween 20 in PBS, washed twice in PBS, and resuspended in 0.5% BSA and PBS (BSA/PBS); immunostaining was then performed using fluorescein-conjugated anticytokeratin antibodies (Ortho, Raritan, NJ) or rabbit antihuman thyroglobulin serum followed by sheep antirabbit IgG as a fluorescein-conjugated secondary antibody. Serum from nonimmunized rabbits or nonspecific fluoresceinated immunoglobulins of the same isotype was used as controls. Cells were then analyzed by flow cytometry using a FACScan apparatus (Becton Dickinson, Mountain View, CA). Monoclonal antibody of mouse origin against ß1-subunit (clone A1A5) was kindly donated by Dr. M. E. Hemler (Dana Farber Cancer Institute, Boston, MA) and anti-3 (J143) by Dr. L. J. Old (Ludwig Institute for Cancer Research, New York, NY). Monoclonal antibody to 5 was purchased from Telios (San Diego, CA); anti-vß3 and anti-vß5 were purchased from Chemicon (Temecula, CA); fluorescein-conjugated antimouse and antirabbit IgG and horseradish peroxidase-conjugated antirabbit IgG were purchased from Ortho. Flow cytometric analysis was performed as follows: cells harvested from subconfluent cell cultures by PBS containing 0.05% trypsin were incubated with the primary monoclonal antibody for 1 h at 4 C in BSA/PBS, washed in the same buffer, and incubated again with the secondary fluorescein-conjugated antibody for 30 min at 4 C. Cells were resuspended in BSA/PBS and analyzed by flow cytometry. Nonspecific immunoglobulins of the same isotype were used as controls. The expression of each integrin was represented as: relative fluorescence index (RFI) = experimental mean fluorescence/control mean fluorescence.
Western blot and immunoprecipitation
For Western blot analysis, the cells were lysed in Laemmli buffer [0.125 mol/liter Tris (pH 6.8), 5% glycerol, 2% sodium dodecyl sulfate (SDS), 1% ß-mercaptoethanol, and 0.006% bromphenol blue], and proteins were resolved by 7–10% SDS-PAGE and transferred to a nitrocellulose membrane (Immobilon P; Millipore Corp., Bedford, MA). Membranes were blocked by 5% nonfat dry milk, 1% ovalbumin, 5% FCS, and 7.5% glycine in PBS, washed, and incubated for 1 h at 4 C with primary antibodies and then washed again and incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were detected by an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). Computer-acquired images were quantified using ImageQuant software (Amersham Biosciences). For immunoprecipitation, the cells were lysed in immunoprecipitation buffer [0.05 mol/liter Tris-HCl (pH 8.0), 0.005 mol/liter EDTA, 0.15 mol/liter NaCl, 1% Nonidet P-40, 0.5% sodium deoxycolate, 0.1% SDS, 0.01 mol/liter NaF, 0.005 mol/liter EGTA, 0.01 mol/liter sodium pyrophosphate, and 0.001 mol/liter phenylmethylsulfonylfluoride]. Rabbit polyclonal antibody reactive to all CaMKII isoforms (Santa Cruz Biotechnology, Santa Cruz, CA) and protein G plus/protein A agarose beads (Oncogene Science, Boston, MA) were used to immunoprecipitate CaMKII from 1 mg of total lysate. Raf-1 and BRaf antibodies were from Santa Cruz Biotechnology. Mouse monoclonal antibodies to Erk-2 and phospho-Erk-1/2 were from Santa Cruz Biotechnology. Polyclonal anti-phospho-CaMKII antibody (pT286-CaMKII) was from Promega (Madison, WI).
p21Ras activity assay
Ras activity was assayed by affinity precipitation using a Ras activation assay kit (Upstate Biotechnology, Lake Placid, NY). Briefly, 4 x 106 cells were lysed with Mg2+ lysis buffer [0.125 mol/liter HEPES (pH 7.5), 0.75 mol/liter NaCl, 5% Igepal CA630, 0.05 mol/liter MgCl2, 0.005 mol/liter EDTA, and 10% glycerol] and incubated with 5 µl of a 50% slurry of Raf-1 Ras binding domain peptide for 30 min at 4 C. The beads were then boiled in reducing sample buffer, and adsorbed proteins were resolved by electrophoresis, transferred to nitrocellulose, and probed with a monoclonal anti-Ras (1 µg/ml) (Mg2+ lysis buffer, Raf-1 Ras binding domain peptide, and anti-Ras were included in a Ras activation assay kit; Upstate Biotechnology, Lake Placid, NY). Proteins were visualized using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence.
Calcium measurement
A total of 3 x 105 cells harvested by trypsin were loaded with cell-permeant fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) by incubating the cells with DMEM, 10–5 mol/liter fura-2, 0.5% BSA, and 0.01 mol/liter HEPES for 30 min at 37 C. The cells were then washed twice for 10 min with 0.001 mol/liter CaCl2 in Hanks’ balanced salt solution [0.118 mol/liter NaCl, 0.0046 mol/liter KCl, 0.01 mol/liter glucose, and 0.02 mol/liter HEPES (pH 7.2)]. When indicated, cells were incubated with integrin-FN-binding arginine-glycine-aspartic acid (RGD)-containing peptides (Gly-Arg-Gly-Asp-Ser-Pro) or control peptides RGE (Gly-Arg-Gly-Glu-Ser-Pro) (Calbiochem, EMD Biosciences, Darmstadt, Germany). Fluorescence was measured with a fluorometer (PerkinElmer Life Sciences, Norwalk, CT). Excitation was at 345 and 380 nm and emission was at 510 nm. Minimal and maximal relative fluorescences were obtained by adding 0.01 mol/liter EDTA and 2% Triton X-100 or 0.01 mol/liter EDTA, 2% Triton X-100, and 0.01 mol/liter CaCl2, respectively. The nanomolar concentration of Ca2+ was obtained by the Grynkiewiez formula considering 225 Kd for fura-2 (15).
[3H]Thymidine incorporation
To determine DNA synthesis, cells were plated in F-12 , 0.5% BSA, and 0.5 µCi [3H]thymidine in 24-well plates coated with FN. After 24 h, the plates were gently washed with PBS avoiding cell loss and then with 10% trichloroacetic acid (TCA) and incubated 10 min with 20% TCA at 4 C. TCA was removed and cells were lysed with 0.2% SDS for 15 min at 4 C. Lysates were then resuspended in 5 ml scintillation fluid and counted in a ß-counter (Beckton Dickinson).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Follicular cells from normal glands obtained by collagenase digestion were cultured for a maximum of 7 d in vitro. The expression of the FN receptors of the integrin family was evaluated by flow cytometry with monoclonal antibodies specific for ß1-, 3, and 5-chains and for vß3 and vß5 heterodimers. Figure 1
reports the average expression of integrin subunits and heterodimers measured in four different cultures at 75% confluence. Analysis was performed on 4- to 5-d-old cultures after two passages. As previously demonstrated for 3ß1 (16), the level of vß3 expression also changed during the culture under the regulatory effect of cell-to-cell contact. The expression of these integrins was higher at 75% confluence than at full confluence. For this reason all experiments were performed on cells after two passages from 4- to 5-d-old cultures at 75% confluence. The ß1- and 3-chains that associate to constitute the 3ß1 etherodimer were strongly expressed as previously demonstrated in normal thyroid cells in culture as well as in tissue sections (4). The vß3 receptor was expressed as previously reported in the thyroid cell line TAD-2 (12), whereas 5 and vß5 were not expressed. Thus, normal thyroid cells in primary culture express only two integrin receptors for FN: 3ß1 and vß3.
|
Thyroid cells were starved from serum for 24 h, harvested, and plated onto FN- or BSA-coated plates. Active p21Ras and phosphorylated Erk were determined in cell extracts (Fig. 2, A and B). Strong p21Ras activation was achieved 15 min after plating the cells onto immobilized FN. Whereas comparable p21Ras activation was observed at 15 and 30 min, induction of Erk phosphorylation was slower and 30 min stimulation was required to induce a powerful phosphorylation. Also cell proliferation was influenced by FN. Cell number was calculated after 24 and 48 h culture onto FN-coated plates in the absence of serum (Fig. 2C). Whereas in the absence of FN and serum cell adhesion was denied, FN alone was sufficient to allow cell adhesion and spreading. FN induced a 60% increase of cell number after 48 h, whereas the in the presence of the Mek inhibitor PD98052, cell proliferation was completely abrogated. The number of cells plated onto BSA-coated plates was not calculated because cell adhesion was very weak in the absence of serum or FN and most of the cells were detached by 48 h culture.
|
To determine which integrin/s generate/s the Ras/Erk pathway, p21Ras activity was determined in cells plated onto FN in the presence of binding-inhibiting antibodies (Fig. 3). An anti-vß3 binding-inhibiting antibody strongly inhibited p21Ras activation induced by FN, whereas anti-3 was ineffective. Inhibition of p21Ras activation by anti-vß3 antibodies was not complete. Although this could be explained by incomplete inhibition of FN-vß3 binding, participation of other factors (other adhesion molecules or FN contaminants) in the p21Ras activation is possible. These data suggest that the integrin vß3 participates in the activation of the Ras/Erk signal pathway in normal human thyroid cells.
|
vß3 induces increase of intracellular calcium concentration
To test whether integrin activation by FN also generates a calcium signal, intracellular free calcium concentration ([Ca2+]i) was measured by fluorometric analysis in cells in suspension in response to soluble FN binding (Fig. 4). Cells were incubated with soluble FN in the presence of peptides inhibiting the integrin-FN binding (RGD) or control peptides (RGE), anti-vß3, or nonrelevant antibodies. After 30 min of suspension, FN plus peptides or antibodies were added to the cells. In the cells treated with FN alone or FN plus control peptide (not shown), [Ca2+]i increased, reaching a 4-fold increase by 60 min. The FN receptor antagonist peptide RGD completely inhibited the [Ca2+]i increase, demonstrating that FN effect was mediated by integrins. The anti-vß3 antibody inhibited the [Ca2+]i increase induced by FN, whereas a control antibody was ineffective. These data suggest that FN activates a Ca2+ signal through the integrin vß3.
|
To determine whether the FN-induced [Ca2+]i increase could activate CaMK enzymatic activity, we evaluated the phosphorylation level of CaMKII (Fig. 5). CaMKII autophosphorylation at T286 residue indicates CaMKII activation. Serum-starved cells were plated onto FN or BSA, and CaMKII T286-phosphorylation was evaluated by Western blot. In the absence of FN no signal was visible by antiphosphothreonine CaMKII antibody at any time. After 15 min of FN stimulation, a sharp band that increased by 30 min stimulation was visible, thus demonstrating that cell binding to FN induces CaMKII activation in thyroid cells.
|
We previously demonstrated that in an immortalized thyroid cell line, Erk phosphorylation induced by FN requires both Ras/Raf-1/Mek and Ca2+/CaMKII signals. To investigate whether this is a general mechanism or it is restricted to that specific cell type, we tested the effects of Ca2+/CaMKII pathway inhibitors on the phosphorylation of Erk after FN binding. To perform this analysis, we used the CaM inhibitors (W7 and TFP), the nonisoform specific CaMK inhibitor (KN93), and a CaMKII-specific inhibitor (ant-CaNtide). This short peptide is derived from the endogenous CaMKII inhibitor-protein CaMKIIN and was made cell permeable by the Antennapedia N-terminal sequence. The cells were plated onto immobilized FN with different inhibitors and the level of Erk-1/2 phosphorylation was evaluated by Western blot (Fig. 6). Erk-1/2 phosphorylation induced by FN was higher than that induced by 10% FCS. KN93, W7, and TFP displayed a dose-dependent inhibitory effect on Erk-1/2 phosphorylation. Ant-CaNtide abolished FN-induced Erk-1/2 phosphorylation at a 5 µM concentration. These data demonstrate that Erk phosphorylation is dependent on CaMKII activation. Thus, the requirement of Ca2+/CaMKII signals in the FN-induced Erk activation is not a feature restricted to a cell line but is a general mechanism in thyroid cells.
|
To determine whether the interaction of Ras/Raf-1/Mek and Ca2+/CaMKII signals occurs at the Raf-1 level, we tested whether CaMKII formed with Raf-1 a multiprotein complex. CaMKII was immunoprecipitated in extracts from cells plated onto BSA or FN. Immunoprecipitated CaMKII and coprecipitated Raf-1 were detected by Western blot by specific antibodies (Fig. 7). Coimmunoprecipitated Raf-1 was visible only in cells stimulated with FN. The CaMK inhibitor KN93 and the CaMKII inhibitor ant-CaNtide completely inhibited the FN-induced Raf-1/CaMKII association. This experiment suggests that FN induces Raf-1 and CaMKII to form a protein complex that depends on CaMKII activation.
|
BRaf overexpression obtained by transfection in human embryonic kidney cells suggested that Ras induces the Raf-1/Braf heterodimerization through the exposure of 14–3-3 binding sites in the COOH terminus of Raf-1 (17). Thus, BRaf heterodimerized with Raf-1 may cooperate with Raf-1 in cellular response to FN. BRaf appears largely expressed in thyroid cells analyzed by Western blot (Fig. 8A). To determine whether BRaf heterodimerized with Raf-1 in the CaMKII/Raf-1 complex induced by FN, the cells were plated onto FN, and CaMKII was immunoprecipitated. Coprecipitated Raf-1 and BRaf were detected by Western blot by specific antibodies (Fig. 8B). Whereas Raf-1 was evident in FN-induced CaMKII coprecipitate, a band for BRaf was barely visible. These results do not support a significant role for BRaf in the signal generated by FN-integrin binding in thyroid cells.
|
[3H]thymidine incorporation was used to measure the DNA synthesis in thyroid cells cultured in serum-free medium on immobilized FN or untreated wells for 24 h (Fig. 9). In the absence of FN, cells were weakly attached to the plates, round shaped, and spreading was limited by 24 h. In FN-coated wells, the cells were well adherent and flat shaped. The presence of inhibitors did not change the attachment, shape, or spreading. The FN stimulation induced 66% increase of [3H]thymidine incorporation. Both KN93 and ant-CaNtide completely abolished such stimulation, demonstrating that FN-induced DNA synthesis requires CaMKII activity.
|
The surface expression of integrin receptors for FN was determined in a cell line originated from normal thyroid (TAD-2), and cell lines originated from papillary carcinomas (NPA and TPC-1). Although thyroid cancer cells have a broad pattern of integrin expression (18), within FN receptors only quantitative changes of 3ß1 and vß3 were found, and de novo expression of vß5 was not observed (Fig. 10A). Because integrin expression is regulated by several factors, data in cultured cells must be confirmed by tissue analysis. Immunohistochemistry with anti-vß3 antibodies confirmed altered expression of this integrin in papillary carcinomas (Fig. 10B). As for cell lines, the level of vß3 expression was demonstrated to be variable. To evaluate the signal response to FN, the cells were plated onto immobilized FN with or without CaMK inhibitor, and the level of Erk-1/2 phosphorylation was evaluated by Western blot (Fig. 9B
). Erk-1/2 phosphorylation was induced in TAD-2 cells and inhibited by KN93 as previously demonstrated (11). In unstimulated papillary carcinoma cell lines, Erk-1/2 was phosphorylated and FN stimulation did not produce any change. CaMKII inhibition by KN93 decreased Erk-1/2 phosphorylation in TPC-1 cells, whereas it was ineffective in NPA cells. The explanation for the insensitivity to CaMK inhibition in NPA cells lies in the presence of BRafVal599 oncogene (19) that is independent from CaMKII activation (our unpublished data).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Within integrins, the two FN receptors, 5ß1 and vß3, are recognized to be important for generating signals that drive proliferation, survival, and spreading (20, 21, 22, 23). Previous studies demonstrated that thyroid cells do not express 5ß1 or vß5, whereas they express vß3 and minimal vß1 in certain conditioned medium (24, 25). Within the FN receptors expressed in thyroid cells, 3ß1 is likely to play only a structural role because its signaling capacity is not yet established, whereas vß3 has a recognized signaling function (8, 26, 27, 28). The complexity and tissue specificity of signaling generated in response to integrin engagement is made possible by the complexity of interactions between multiple factors that take place in the focal adhesion. In response to 5ß1 and vb3 engagement, the Ras/MAPK signal pathway is activated through the adapter molecules Shc and Grb2/Sos involving FAK, paxillin, or c-Srk in fibroblasts and epithelial and endothelial cells (29, 30, 31). Thyroid cells cultured in vitro in medium also containing hypothalamus and pituitary extracts express vß3 integrin in a latent state characterized by its inability to cluster at focal adhesions and promote cell adhesion. However, vß3 recruitment at focal adhesions and ligand-binding activity occurred on treatment with hepatocyte growth factor/scatter factor (25). In the normal thyroid cell line TAD-2, vß3 membrane expression is restricted to focal adhesions, whereas in the present study, in normal thyroid cells cultured in the absence of growth factors, vß3 stained by immunofluorescence displayed a fine grainy pattern of distribution (not shown). This membrane localization not restricted to focal adhesions suggests the possibility that the effect measured in our system might be of a higher magnitude in a more physiological environment in the presence of those endocrine and paracrine factors abundant in vivo. All together these data indicate that culture conditions and growth factors are relevant modulators of expression and function of integrins in thyroid cells and that normal thyroid cells potentially express active vß3 integrin.
Cell proliferation is regulated by converging signals on the cell cycle machinery that determine whether the cell stays in the G1 phase or proceeds to S phase. The progression through G1 into the DNA synthesizing S phase is driven by cyclin-dependent kinase (CDK)4 and CDK6, which interact with the cyclin D family of proteins, and CDK2, which interacts with cyclins A/E (32). The Ras/Raf/Mek/Erk cascade plays a pivotal role in the control of this process. Sustained Erk activation is required to pass the G1 restriction point and regulate cyclin D1 expression during mid-G1 phase (33). Inhibition of FN-stimulated proliferation by Mek inhibitor demonstrates the role of Erk in this process in thyroid cells. In normal cells, it is unlikely that integrin might sustain prolonged proliferation in the absence of other growth factors. This topic may not be investigated in primary cultures of thyroid cells because they have a very limited growth potential. Our results demonstrate the involvement of vß3 and the Ras/Erk signal in FN-stimulated proliferation in culture, whereas in vivo the entire pathway or part of it must be abrogated or counteracted. Integrins are constantly engaged by the ECM of the basal lamina surrounding the thyroid follicles. Thus, whereas the PI-3K signal might be continuous and relevant in survival, the Ras/Erk signal must be downmodulated to avoid aberrant stimulation of proliferation. Thyroid-stimulating hormone that has been proposed to inhibit Ras signaling to Raf/MAPK might represent one of these factors (34, 35).
Although this and other factors might be responsible of such a regulation, the complexity of signals generated by integrins makes possible their self-modulation. Within signals generated by integrins, PI-3K/Cdc42/Pak represents a costimulatory signal that modulates the Ras/Erk pathway. In COS-7 cells, PI-3K inhibition induces severe attenuation of integrin-dependent Erk activation (36). Integrin-FN binding activates the serine/threonine kinase Pak through PI-3K and Cdc42. Pak then phosphorylates Raf-1 on Ser338, ensuing Erk activation by Ras (36, 37). Whereas Ser338-Raf-1 phosphorylation is necessary for Raf-1 to be fully activated by Ras (38, 39), integrins are not the sole activators of Pak-1, and in any system Pak-1 is not always necessary for Raf-1 activation, possibly because it is substituted by other factors. Indeed, Pak-1 inhibition in Rat-1 fibroblasts abrogates Ras-induced transformation, whereas it is ineffective in NIH-3T3 fibroblasts in which other factors must coactivate Raf-1 (40, 41). Also in TAD-2 cells, FN binding induces a modest level of Pak-1 phosphorylation whose role was not investigated (11). In TAD-2 cells and primary thyroid cultures, besides the Ras/Erk signal, integrin engagement activates the Ca2+/CaMKII signal. CaMKII and Ras are both necessary to activate Raf-1, and inhibition of CaMKII abrogates Erk phosphorylation and proliferation induced by FN. Thus, integrins can self-modulate their own proliferation signal through the PI-3K/Pak-1 and Ca2+/CaMKII signals. Coimmunoprecipitation experiments demonstrate that CaMKII and Raf-1 participate in a common multiprotein complex. The question whether Raf-1 is a direct CaMKII substrate was not addressed in this study. However, this is a possibility worthy of consideration because Raf-1 is a substrate for many kinases and presents a number of consensus sequences for CaMKII.
The malignant behavior of cancer cells is determined by not simply uncontrolled growth but also the ability to migrate, proliferate, and survive in denied adhesion or ectopic body districts. Tumor cells metastasized into ectopic sites or moving in the vascular or lymphatic system loose adhesion to the basal lamina mediated by integrins. The adhesion receptors expressed on the surface of invading cells play a fundamental role in this process. Changes in the level of surface expression of integrins or de novo expression or activation state occur in a number of neoplastic lesions including thyroid cancer (18, 42, 43, 44, 45, 46). The two thyroid cancer cell lines we examined in this study displayed aberrant integrin expression, and Erk resulted in a phosphorylated state also in denied adhesion. Independently whether Erk activation resulted from activated integrins, oncogenes, or other activating pathways, aberrant integrin expression and activation might still play a relevant role as a costimulatory factor. In TPC-1 cells the Ca2+/CaMKII signal is still necessary to Erk phosphorylation. In this cell line harboring RET/PTC-1, this oncogene might be responsible of sustained Erk phosphorylation, and CaMKII can be activated by integrins or other effectors including RET/PTC-1 oncogene. Conversely, tumor cells harboring oncogenes that do not lie on the tyrosine kinase/Raf-1 pathway elude the Raf-1/CaMKII checkpoint. The mutation at site T1796 is sufficient to generate a BRaf mutant constitutively activated (47). One relevant difference between Raf-1 and BRaf is the presence of the S445 in BRaf. This serine is equivalent to S338 of Raf-1 and is constitutively phosphorylated (39). Also for this reason, Ras alone is sufficient to activate BRaf and Val599E mutation activates BRaf with no need for other factors. A number of biological consequences with clinical relevance arise from the Raf-1/CaMKII interaction. Future studies must determine the factors responsible for CaMKII activation in tumors. Besides altered integrins and known physiological factors, mutated Ras, Trk, and RET/PTC have the potentiality to autonomously activate the Ca2+/CaMKII signal. A different ability to activate CaMKII might be responsible for the different aggressive behavior observed within RET/PTC mutants and different oncogenes.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
First Published Online February 1, 2005
Abbreviations: BSA/PBS, 0.5% BSA and PBS; Ca2+, calcium; [Ca2+]i, intracellular free calcium concentration; CaMKII, calcium calmodulin-dependent kinase II; CDK, cyclin-dependent kinase; ECM, extracellular matrix; F-12, Ham’s F-12 medium; Fak, focal adhesion kinase; FCS, fetal calf serum; FN, fibronectin; Mek, MAPK kinase; PI-3K, phosphatidylinositol-3 kinase; PTC, papillary thyroid carcinoma; RFI, relative fluorescence index; RGD, arginine-glycine-aspartic acid; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.
Received July 31, 2004.
Accepted January 20, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
5ß1 integrin receptor in the proliferative response of quiescent human melanoma cells to fibronectin. Cancer Res 52:4499–4506vß6 integrin promotes proliferation of colon carcinoma cells through a unique region of the ß6 cytoplasmic domain. J Cell Biol 127:547–556vß3 integrin in normal epithelial cells: implications for tumor invasion. J Cell Biol 142:1145–11562ß1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis. J Cell Sci 108(Pt 11):3531–3540
This article has been cited by other articles:
 |
|
 |
|
  M. R. Rusciano, M. Salzano, S. Monaco, M. R. Sapio, M. Illario, V. De Falco, M. Santoro, P. Campiglia, L. Pastore, G. Fenzi, et al. The Ca2+-calmodulin-dependent kinase II is activated in papillary thyroid carcinoma (PTC) and mediates cell proliferation stimulated by RET/PTC Endocr. Relat. Cancer, January 29, 2010; 17(1): 113 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lorger, J. S. Krueger, M. O'Neal, K. Staflin, and B. Felding-Habermann Activation of tumor cell integrin {alpha}v{beta}3 controls angiogenesis and metastatic growth in the brain PNAS, June 30, 2009; 106(26): 10666 - 10671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Li, C. Wang, Y. Wu, X. Liu, and X. Cao Ca2+/Calmodulin-dependent Protein Kinase II Promotes Cell Cycle Progression by Directly Activating MEK1 and Subsequently Modulating p27 Phosphorylation J. Biol. Chem., January 30, 2009; 284(5): 3021 - 3027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Macias-Perez, C. Borza, X. Chen, X. Yan, R. Ibanez, G. Mernaugh, L. M. Matrisian, R. Zent, and A. Pozzi Loss of Integrin {alpha}1{beta}1 Ameliorates Kras-Induced Lung Cancer Cancer Res., August 1, 2008; 68(15): 6127 - 6135. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Wang, N. Li, X. Liu, Y. Zheng, and X. Cao A Novel Endogenous Human CaMKII Inhibitory Protein Suppresses Tumor Growth by Inducing Cell Cycle Arrest via p27 Stabilization J. Biol. Chem., April 25, 2008; 283(17): 11565 - 11574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sanhueza, C. C. McIntyre, and J. E. Lisman Reversal of Synaptic Memory by Ca2+/Calmodulin-Dependent Protein Kinase II Inhibitor J. Neurosci., May 9, 2007; 27(19): 5190 - 5199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Han, J. D. Ritzenthaler, B. Wingerd, H. N. Rivera, and J. Roman Extracellular Matrix Fibronectin Increases Prostaglandin E2 Receptor Subtype EP4 in Lung Carcinoma Cells through Multiple Signaling Pathways: THE ROLE OF AP-2 J. Biol. Chem., March 16, 2007; 282(11): 7961 - 7972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Wang, K. Peyrollier, and L. Y. Bourguignon The Influence of Hyaluronan-CD44 Interaction on Topoisomerase II Activity and Etoposide Cytotoxicity in Head and Neck Cancer Arch Otolaryngol Head Neck Surg, March 1, 2007; 133(3): 281 - 288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Paternot, J. E. Dumont, and P. P. Roger Differential Utilization of Cyclin D1 and Cyclin D3 in the Distinct Mitogenic Stimulations by Growth Factors and TSH of Human Thyrocytes in Primary Culture Mol. Endocrinol., December 1, 2006; 20(12): 3279 - 3292. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Han, J. D. Ritzenthaler, S. V. Sitaraman, and J. Roman Fibronectin Increases Matrix Metalloproteinase 9 Expression through Activation of c-Fos via Extracellular-regulated Kinase and Phosphatidylinositol 3-Kinase Pathways in Human Lung Carcinoma Cells J. Biol. Chem., October 6, 2006; 281(40): 29614 - 29624. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Guarino, P. Faviana, G. Salvatore, M. D. Castellone, A. M. Cirafici, V. De Falco, A. Celetti, R. Giannini, F. Basolo, R. M. Melillo, et al. Osteopontin Is Overexpressed in Human Papillary Thyroid Carcinomas and Enhances Thyroid Carcinoma Cell Invasiveness J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5270 - 5278. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
