Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Texas 78712
Submitted 27 June 2003 ; accepted in final form 25 October 2003
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ABSTRACT |
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cell cycle extracellular signal-regulated kinase; quinol-thioether reactive oxygen species
The mechanism underlying the antiproliferative function of tuberin is still relatively uncharacterized. Recent studies showed that the Akt pathway regulates the formation of the tuberous sclerosis complex (7). Components of this complex, in turn, inhibit signaling mediated by the mammalian target of rapamycin (28). Tuberin is a structurally complex protein, containing several functional domains (10, 30) including a region homologous to a portion of the catalytic domain of the GTPase-activating protein (GAP) for Rap1 (35). As predicted by sequence homology, tuberin exhibits modest GAP activity toward Rap1 (35). Moreover, tuberin colocalizes with Rap1 in the Golgi apparatus (37) and both tuberin and Rap1 have similar patterns of tissue expression, including expression in the kidney (36). Although there is variability in the localization of immunoreactive tuberin in small blood vessels of many human organs, including the kidney, skin, and adrenal glands (3), the ability of tuberin to act as a GAP toward Rap1, and the coexpression of tuberin and Rap1, provides strong circumstantial evidence for a physiological role for tuberin in the regulation of Rap1. Rap1 is a member of the Ras superfamily of GTP-binding proteins and shares 50% amino acid sequence identity with Ras (31). The similarity with the effector domain of Ras is striking, and Rap1A has an identical effector domain to that of Ras (17) and associates with almost all cellular effectors of Ras, including Raf-1 (20) and B-Raf (32). Like Ras, Rap1 has been implicated in a variety of cellular processes, including cell proliferation and differentiation (41). Although GAP activity appears to be important for the antitumorigenic effects of tuberin (9, 35), the physiological significance of tuberin on Rap1 function is still unknown.
The Tsc-2 tumor suppressor gene is an early target of reactive oxygen species (ROS)-induced nephrocarcinogenicity (11). Loss of heterozygosity (LOH) of the Tsc-2 gene followed by loss of tuberin expression occurs in quinol-thioether-induced renal tumors (11). Quinol-thioether-transformed rat renal epithelial (QT-RRE) cell lines were subsequently established from primary renal epithelial cells treated with 2,3,5-(trisglutathion-S-yl)hydroquinone, and these cells lack tuberin expression due to LOH of the Tsc-2 gene (40). The expression of ERK activity is elevated in both ROS-induced renal tumors and QT-RRE cells (39). Interestingly, restoration of tuberin expression in QT-RRE cells decreases ERK activity, suggesting that loss of tuberin expression is coupled to high ERK activity (39). ERK is a member of the MAPK superfamily that play central roles in many cellular processes, including cell proliferation, differentiation, and oncogenic transformation, dependent on cellular context (18). ERK may also be involved in the development of renal tumors and their malignancy (5, 21). These findings suggest that the Tsc-2 gene might exert its tumor suppressor function via the ERK-signaling pathway.
In the present study, we sought to identify possible downstream mediators regulated by tuberin, using QT-RRE cells transfected with Tsc-2 cDNA to restore tuberin expression. The high ERK activity observed in QT-RRE cells was not due to constitutive activation of the EGF receptor (EGF-R), which is a common phenomenon in the majority of renal cancers. Restoration of tuberin expression in QT-RRE cells by transient transfection with Tsc-2 cDNA significantly decreased both ERK and B-Raf activity. Thus our study provides some insights into the mechanisms by which tuberin exerts its tumor-suppressive action.
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MATERIALS AND METHODS |
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Cell cultures. Three independent tuberin-negative cell lines, QT-RRE1, 2, and 3, were established from primary renal epithelial cells (40). The cells were grown in medium consisting of 50% DMEM and 50% Ham's F-12 with 10% FBS. LLC-PK1 (porcine proximal tubule epithelial cells) and NRK-52E (normal rat kidney epithelial cells) were from American Type Culture Collection (Manassas, VA) and were maintained in DMEM with 10% FBS or 10% calf serum, respectively. All cell lines were grown at 37°C in a humidified atmosphere of 5% CO2.
Transient transfection. A full-length Tsc-2 cDNA in the pcDNA3 expression vector was kindly provided by Dr. R. S. Yeung (University of Washington, Seattle, WA). Transient transfection with 4 µg of Tsc-2 cDNA was performed with QT-RRE cells between 60 and 90% confluence in either six-well or 100-mm plates using LipofectAMINE or LipofectAMINE Plus according to the manufacturer's protocol. For control, QT-RRE cells were transfected with control vector pcDNA3 (Invitrogen). The transfected cells were harvested 36 to 48 h after transfection. Tuberin expression in the transfected cells was confirmed by Western blot analysis.
3[H]thymidine incorporation. QT-RRE cells were seeded at a density of 105 cells/well in six-well plates. At 60% confluence, transient transfection with either Tsc-2 cDNA or vector pcDNA3 was performed as described above. Thirty-six hours following transfection, cells were incubated with 5 µCi/ml of 3[H]thymidine for 4 h. After the incubation, the cells were washed three times with PBS, pH 7.4, and 5% TCA was added. Cells were harvested by gentle scraping and centrifuged for 15 min at 14,000 rpm. The supernatant was removed, and the cell pellets were washed three times by adding 5% TCA to remove unincorporated 3[H]thymidine. A final washing was performed with absolute alcohol to remove residual TCA, and the pellets were dried. Dried pellets were dissolved in 1 ml of 0.1 N NaOH. Radioactivity was determined by liquid scintillation spectroscopy (Beckman LS5000TD), and protein concentrations were measured with the Bradford method.
Measurement of ERK activity. Total MAPKp44/p42 (ERK1/ERK2) or MAPK p44 and p42 activity were determined with immunoprecipitated protein or with a p42/p44MAP kinase enzyme assay kit. Cells were homogenized and lysed with lysis buffer containing 50 mM Tris·HCl (pH 7.5), 100 mM NaCl, 10 mM sodium fluoride, 5 mM EDTA, 1% Triton X-100, 40 mM -glycerophosphate, 0.5 mM sodium orthovanadate, 0.25 mM PMSF, and protease inhibitors. Cell lysates were cleared by centrifugation, and endogenous MAPKp44/p42 was immunoprecipitated from 500 µg of total protein from the lysates using a goat anti-ERK1 polyclonal antibody C-16 from Santa Cruz Biotechnology (Santa Cruz, CA) followed by protein G-Sepharose (Santa Cruz Biotechnology). The immunoprecipitates were washed extensively with PBS and assayed for kinase activity using MBP as a substrate. The incorporated [
-33P]ATP or [
-32P]ATP was quantified by liquid scintillation spectroscopy.
Measurement of B-Raf and Raf-1 kinase activity. Kinase activity was determined using B-Raf or Raf-1 immunoprecipitation kinase cascade kits (Upstate Biotechnology) according to the manufacturer's instructions. Cells were lysed with buffer A containing 50 mM Tris·HCl (pH 7.5), 100 mM NaCl, 10 mM sodium fluoride, 5 mM EDTA, 1% Triton X-100, 40 mM -glycerophosphate, 0.5 mM sodium orthovanadate, 0.25 mM PMSF and protease inhibitors. Total protein (500 µg) was immunoprecipitated using a sheep anti-B-Raf or Raf-1 polyclonal antibody (Upstate Biotechnology) bound to protein G-agarose beads (Santa Cruz Biotechnology). The protein G-agarose/enzyme-immunocomplex was washed twice with 500 µl of ice-cold buffer A and suspended with 80 µl assay dilution buffer (ADB) containing 20 mM MOPS, pH 7.2, 25 mM
-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol. For each Raf-dependent activation of inactive GST-p42MAPK, 20 µl of ADB, 10 µl of magnesium/ATP cocktail (500 µM ATP and 75 mM MgCl2 in ADB), 0.4 µg of inactive MEK1, and 1 µg of inactive MAPK2/ERK2 were incubated with the protein G-agarose/enzyme-immunocomplex for 30 min at 30°C in a shaking incubator. After centrifugation to pellet the agarose beads, 4 µl of the supernatant were used for the second-stage reaction of phosphorylation of MBP by activated MAPK2/ERK2. For the second-stage reaction, 10 µl of ADB, 20 µg of MBP substrate, and 10 µCi of [
-33P]ATP mixture were incubated with 4 µl of activated MAPK2 for 15 min at 30°C in a shaking incubator. The reaction mixture (25 µl) was spotted onto a phosphocellulose membrane. The membranes were washed with 0.75% phosphoric acid, and the incorporated [
-33P]ATP was quantified by liquid scintillation spectroscopy.
RT-PCR determination of B-Raf. Total RNA was isolated by the guanidinium isothiocyanate/phenol extraction method, and 1 µg was reverse transcribed using a Retroscript kit from Ambion (Austin, TX). The reverse-transcribed product (5 µl) was used for PCR amplification in a DNA thermocycler programmed to cycle at 95°C for 1 min, 54°C for 1 min, and 72°C for 1 min for 40 cycles, followed by incubation at 72°C for 5 min. Amplification was performed in a volume of 50 µl, containing 10 mM Tris·HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl, 0.25 mM dNTP, 2.5 U of Taq DNA polymerase from Roche Diagnostic (Indianapolis, IN), and 25 pmol each of sense and antisense primers. The primers used to amplify 513 bp of the B-raf gene were BRAF1 5'-CACGCCAAGTCAATCATCC-3'(forward) and BRAF1 5'-GAAACCAGCCCGATTCAAG-3' (reverse). After amplification, the PCR products were electrophoresed on a 1.5% agarose gel along with size markers.
Western blot analysis. QT-RRE cells were homogenized with lysis buffer (1x PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS containing PMSF, aprotinin, and sodium orthovanadate). Cell debris was cleared by centrifugation at 14,000 g for 30 min at 4°C. Total protein (100 µg for tuberin and cyclin D1 and 50 µg for ERK1/2) was subjected to SDS-polyacrylamide gel electrophoresis (7% resolving for tuberin and 12% resolving for all other proteins). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes at 100 V (3 h for tuberin and 1 h for all other proteins). The PVDF membranes were blocked in 5% nonfat dry milk in TBS-T buffer [25 mM Tris·HCl, pH 7.6; 0.2 mM NaCl; 0.1% Tween 20 (vol/vol)] for 1 h at room temperature and then incubated with the respective primary antibodies [tuberin (C-20), cyclin D1 (A-12)] from Santa Cruz Biotechnology, actin (for -,
-, and
-isoforms; AB-1 kit) from Oncogene Research Products (Boston, MA), and ERK1/2 (9102) from Cell Signaling (Beverly, MA) at a dilution of 1:100 for tuberin and 1:500 for all other proteins overnight at 4°C. After being washed with TBS-T, the membranes were incubated with the corresponding secondary antibody at a dilution of 1:2,000. After being washed, membranes were visualized by ECL. The membranes were stripped with 0.2 M NaOH for 5 min each, blocked with 5% milk for 15 min, and then incubated with the respective primary and secondary antibodies as described earlier.
Statistics. Data are expressed as means ± SE. Mean values were compared using a two-way ANOVA, followed by Student-Newman-Kuel's test of statistical significance.
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RESULTS |
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QT-RRE cells have lost the remaining functional allele at the Tsc-2 locus and thus do not express tuberin. The restoration of Tsc-2 cDNA into QT-RRE cells significantly decreases ERK activity, demonstrating that ERK activity is negatively regulated by tuberin (39). Genistein, an inhibitor of protein tyrosine kinases (PTK), was used to examine whether upstream tyrosine kinases contribute to the high ERK activity in QT-RRE cells. Genistein (100 µM) significantly decreased ERK activity in QT-RRE cell lines (Fig. 2), suggesting that the constitutive activation of ERK is, in part, due to the activity of upstream tyrosine kinases.
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Constitutive ERK expression in QT-RRE cells is independent of the EGF-R. We hypothesized that the constitutive activation of the ERK cascade in QT-RRE cells might arise via constitutive activation of the EGF-R, an effect frequently observed in renal cancer, rather than via any direct effects of deregulated tuberin expression. This possibility was investigated by measuring ERK activity in QT-RRE cells treated with a specific EGF-R inhibitor, AG-1478. AG-1478 (200 nM) had no effects on ERK activity in QT-RRE cells (Fig. 3). In contrast, AG-1478 caused a significant decrease in ERK activity in NRK-52E cells, a commercially available cell line that expresses constitutively high ERK activity. Stimulation of the EGF-R with EGF in the QT-RRE cells demonstrated that these cells possess a functional EGF/EGF-R/ERK-signaling pathway. However, our data demonstrate that constitutive activation of EGF-R does not contribute to high ERK activity in QT-RRE cells.
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Inhibition of new DNA synthesis by tuberin. ERK generally mediates cell proliferation. We previously showed that restoration of tuberin expression suppresses ERK activity in QT-RRE cells (39). However, restoration of tuberin expression (Fig. 4A, top) had little effect on total ERK 1 and 2 expression in QT-RRE 1 and 2 (Fig. 4A, middle). There was little change in actin expression (Fig. 4A, bottom) between cells transfected with the empty pcDNA3 vector and cells transfected with pcDNA3 containing Tsc-2 DNA. To investigate whether the lack of tuberin expression causes high proliferative activity in QT-RRE cells, transient transfection with Tsc-2 cDNA was carried out to restore tuberin expression. Tuberin restoration decreased new DNA synthesis in QT-RRE 1 and QT-RRE 2 cell lines by 22 and 45%, respectively (Fig. 4B), which is consistent with the view that decreases in ERK activity subsequent to restoration of tuberin expression contribute to the suppression of cell proliferation.
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Downregulation of cyclin D1 expression by tuberin. Cyclin D1 is a key regulator of G1 progression in the mammalian cell cycle and is overexpressed in several tumor types including renal cell carcinomas (4, 13). We therefore measured the level of expression of cyclin D1 in QT-RRE cells by Western blot analysis. QT-RRE cells express high levels of cyclin D1, which are substantially downregulated by the restoration of tuberin expression (Fig. 5), indicating that regulation of the cell cycle by tuberin is, at least in part, mediated by modulation of cyclin D1. Expression of tuberin again had little effect on actin (housekeeping protein) expression in QT-RRE cells (data not shown).
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High B-Raf activity in QT-RRE cells. Three enzymes, Raf, MEK, and ERK, comprise the ERK-signaling cascade, which is an essential pathway for mitogenic signal transduction in many cell types (19). In particular, Raf kinases serve as central intermediates in many mitogenic pathways and oncogenic transformation. To investigate whether Raf kinase(s) contribute to high ERK activity in QT-RRE cells, B-Raf gene expression was determined by RT-PCR analysis. The B-Raf transcript was expressed in all three QT-RRE cell lines (Fig. 6A). B-Raf and Raf-1 activity were subsequently measured following immunoprecipitation. B-Raf activity was higher in QT-RRE cells compared with LLC-PK1 cells, although activity varied within the three cell lines (Fig. 6B). Restoration of tuberin expression in QT-RRE cells markedly decreased B-Raf activity in all these cell lines (60-70%), indicating that tuberin acts as an upstream regulator of B-Raf activity (Fig. 6C). Levels of Raf-1 in QT-RRE cells were also five- to ninefold higher than those in NRK-52E and LLC-PK1 cells (Fig. 7A). However, restoration of tuberin expression produced only modest decreases in Raf-1 activity (20-25%). Thus tuberin has a greater impact on B-Raf activity than on Raf-1 activity (Fig. 7B).
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DISCUSSION |
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Constitutive activation of ERK in QT-RRE cells could occur as a consequence of deregulation of upstream signaling molecule(s). Genistein, an inhibitor of PTKs, significantly decreased ERK activity in QT-RRE cell lines (Fig. 2), suggesting that upstream tyrosine kinase(s) are involved in ERK activation in these cells. Although the Src family are the major PTKs (genistein is a nonspecific PTK inhibitor), it is unknown at this time which particular PTK contributes to the upregulation of the ERK pathway in QT-RRE cells.
The finding that restoration of tuberin expression in QT-RRE cells substantially decreases both ERK (39) and B-Raf (Fig. 6C) activity establishes the ability of tuberin to behave as a negative regulator of the ERK-signaling pathway. The participation of B-Raf in the activation of the ERK cascade is intriguing, because there are few reports of B-Raf in renal epithelial cells. Similar to other Raf kinases, B-Raf plays an essential role in cell proliferation, development, and cell survival (2). However, the regulation of B-Raf differs from that of Raf-1, the most common and well-studied Raf, in that B-Raf can be activated by both Ras and Rap1, whereas Raf-1 is activated only by Ras (29, 32, 41). B-Raf may be the primary target of oncogenic events involving the three Raf isoforms (15). Expression of B-raf is tissue and cell type specific. Thus B-raf expression in QT-RRE cells was confirmed by RT-PCR (Fig. 6A).
Signal integration is quite complex upstream of B-Raf, because there is input from many other signaling cascades. Ras is a well-characterized upstream effector of the ERK cascade. However, Rap1, a ras-related GTP-binding protein, also lies upstream in the ERK pathway and regulates the B-Raf/ERK cascade (32, 41). Tuberin has been reported to function as a Rap1GAP (35). Of the four Rap1GAPs identified to date, tuberin is the only reported Rap1GAP expressed in the kidney. Tuberin may therefore be the predominant Rap1GAP in the kidney. In this case, loss of tuberin would predispose cells to deregulation of the Rap1-mediated signaling pathway. Sustained activation of Rap1 may therefore represent the focus for the deregulation of the ERK-signaling pathway caused by the loss of tuberin expression. However, the signaling pathway emanating from a lack of tuberin expression leading to stimulation of B-Raf and ERK is presently unclear.
Basal activity of both B-Raf and Raf-1 was enhanced in QT-RRE cells (Figs. 6B and 7A). However, whereas restoration of tuberin in these cells remarkably decreased B-Raf activity (Fig. 6B), Raf-1 activity was only slightly diminished (Fig. 7B), indicating that B-Raf is likely the major Raf kinase regulated by tuberin in QT-RRE cells. Moreover, the basal activity of B-Raf is similar to that of ERK (0.15 ± 0.02 pmol pi·min-1·µg protein-1), which is 10 times higher than Raf 1 (0.014 ± 0.002 pmol pi·min-1·µg protein-1) in QT-RRE cells, suggesting a preferential link for a B-Raf/ERK-signaling pathway. Raf-1 is expressed ubiquitously and is a major immediate downstream effector of Ras (2). Although B-Raf is a close structural homolog of Raf-1, there are notable differences in their modes of regulation (15). Raf-1 is activated by Ras, not by Rap1, whereas B-Raf is activated by both Ras and Rap1. B-Raf is coupled to Rap1, upon which tuberin may act as a negative regulator (32, 35). The finding that B-Raf is markedly downregulated by tuberin in QT-RRE cells is consistent with the view that Rap1 also contributes to the mechanism by which tuberin regulates the ERK cascade. A-Raf is the least-studied kinase in the Raf family and its contribution to the MAPK signaling pathway has yet to be clarified (15). It is interesting to note that a recent study showed that B-Raf/Rap1 signaling, but not c-Raf-1/Ras, induces the histidine decarboxylase promoter in Heliobacter pylori infection (34), indicating that B-raf/Rap1 and Raf-1/Ras are independent upstream activators of the MEK/ERK-signaling cascade.
MEK is the target for convergent regulation by a diverse group of upstream activators and is the only in vivo substrate so far identified that is common to all Raf proteins (16). Similarly, ERK is the only downstream effector of MEK, and constitutive activation of ERK is mostly associated with the constitutive activation of MEK (5). Thus, although MEK activity was not measured in these studies, activation of ERK is likely mediated through B-Raf and MEK in QT-RRE cells. Constitutively active MEK could, therefore, drive cell proliferation in QT-RRE cells, but MEK would be suppressed in the Tsc-2-transfected cells.
QT-RRE cells also exhibit elevated cyclin D1 expression (39). This elevation is likely related to high ERK activity in QT-RRE cells (Fig. 1). Restoration of tuberin expression decreased cyclin D1 expression in QT-RRE cells (Fig. 5), consistent with the previous finding that Tsc-2 antisense oligonucleotides increase cyclin D1 expression (25). Cyclin D1 is a downstream effector of ERK (12), although the molecular events leading to increases in cyclin D1 expression subsequent to ERK activation remain unclear. Our results provide strong evidence coupling tuberin to cell cycle progression. As predicted by cyclin D1 levels, restoration of tuberin expression decreased cell proliferation in QT-RRE cells (Fig. 4). Moreover, these results are in accord with previous reports (9, 23) demonstrating that introduction of the wild-type Tsc-2 gene into tuberin-negative cell lines suppresses cell proliferation. The antiproliferative effects of tuberin in QT-RRE cells could be reduced via the deregulation of the ERK cascade (B-Raf/MEK/ERK/cyclin D1) (Fig. 8). Although our data establish a link between tuberin and downstream molecular signaling networks, additional pathways may contribute to tumor development, and in fact several studies have demonstrated a role for tuberin in cell proliferation (7, 28).
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In summary, we have shown that restoration of tuberin in QT-RRE cells decreases ERK and B-Raf activity and the expression of cyclin D1. The results strongly suggest that tuberin is an upstream negative regulator of the ERK signaling cascade and that loss of tuberin expression in QT-RRE cells disrupts the B-Raf/MEK/ERK/cyclin D1 cascade. The aberrant constitutive activation of the ERK cascade causes cellular transformation and tumor development (5, 6, 14, 21, 22). Our findings are consistent with the view that the Tsc-2 gene exerts its tumor suppressor activity, at least in part, via regulation of the ERK cascade.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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