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【摘要】 The tuberous sclerosis-2 ( Tsc-2 ) gene is a suppressor of renal tumorigenesis and an early target of reactive oxygen species-induced renal cancer. Tuberin, the protein product of the Tsc-2 gene, participates in the regulation of cell proliferation, although the mechanism by which it suppresses proliferation is unknown. Quinol-thioether-transformed rat renal epithelial (QT-RRE) cell lines, derived from quinol-thioether-transformed primary renal epithelial cells from Eker rats, lack tuberin expression due to loss of heterozygosity of the Tsc-2 gene. These cell lines were used to examine the mechanism by which tuberin exerts its antiproliferative action. Loss of tuberin function correlates with high ERK activity ( 39 ), which could contribute to the formation of renal tumors. In this study, we sought to identify possible downstream effectors regulated by tuberin, using QT-RRE cells transfected with Tsc-2 cDNA to restore tuberin expression. Constitutively high ERK, B-Raf, and Raf-1 activities were observed in QT-RRE cells. However, restoration of tuberin expression in QT-RRE cells by transient transfection with Tsc-2 cDNA substantially decreased both ERK and B-Raf activity, with only modest changes in Raf-1 activity, suggesting tuberin functions as an upstream negative regulator of the ERK pathway. High ERK activity was not mediated through EGF receptor activation, but treatment with genistein demonstrated that protein kinases are involved in ERK cascade activation. The data indicate that loss of tuberin results in the upregulation of the ERK signaling pathway with subsequent increases in new DNA synthesis, and ultimately, tumor formation.
【关键词】 cell cycle extracellular signalregulated kinase quinolthioether reactive oxygen species
THE TUBEROUS SCLEROSIS -2 ( Tsc-2 ) gene functions as a renal tumor suppressor ( 33 ), although the mechanism of this effect remains elusive ( 25, 35, 38 ). Tuberin, the Tsc-2 -encoded protein, regulates cell growth and proliferation ( 1, 9, 23, 24, 27 ) and cell cycle progression ( 25, 26 ). Loss of tuberin expression induces quiescent G 0 -arrested cells to enter the cell cycle and prevents cells from reentering a quiescent state ( 25 ). Similarly, a reduction in the level of tuberin increases cyclin D 1 protein expression and promotes the transition from G 0 /G 1 to S phase. Inactivation of gigas, the Drosophila homolog of Tsc-2, results in the formation of tissues composed of enlarged cells with a repeating S phase without entering the M phase ( 8 ).
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.
MATERIALS AND METHODS
Materials. AG-1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline], genistein (4',5,7-trihydroxyisoflavone), and PD-98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] were purchased from Calbiochem (La Jolla, CA). DMEM/Ham's F-12 media, lipofectAMINE, lipofectAMINE Plus, and OPTI-MEM medium were obtained from Life Technologies (Grand Island, NY). The MAPK p44/p42 assay kit was purchased from Amersham Life Science (Piscataway, NJ), and B-Raf and Raf-1 immunoprecipitation kinase cascade kits were products of Upstate Biotechnology (Lake Placid, NY). [ - 33 P]ATP (3,000 Ci/mmol), [ - 32 P]ATP (3,000 Ci/mmol), and 3 thymidine (60 Ci/mmol) were purchased from DuPont-New England Nuclear (Boston, MA). Unless otherwise stated, all other materials were obtained from Sigma (St. Louis, MO).
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-PK 1 (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% CO 2.
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 thymidine incorporation. QT-RRE cells were seeded at a density of 10 5 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 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 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 MAPK p44/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 MAPK p44/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 [ - 33 P]ATP or [ - 32 P]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 MgCl 2 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 [ - 33 P]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 [ - 33 P]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 (1 x 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 D 1 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 D 1 (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.
RESULTS
High ERK activity in QT-RRE cells. QT-RRE cells are devoid of tuberin ( 40 ). We therefore determined the constitutive expression of ERK in these cells and in the tuberin-positive kidney proximal tubule epithelial cell line (LLC-PK 1 ). To avoid growth stimulation in normal media containing 10% FBS, all the cells were maintained in low-serum media, containing 2% FBS, for 2 days before ERK 1 and 2 activity was measured. QT-RRE cells express approximately fourfold higher basal ERK activity than LLC-PK 1 cells ( Fig. 1 A ), suggesting that ERK 1 and 2 activity is constitutively upregulated in QT-RRE cells. PD-98059 (50 µM), a MEK1/2 inhibitor, was used to confirm the specificity of ERK activity in QT-RRE cells ( Fig. 1 A ). Consistent with the ERK activity assays, Western immunoblotting demonstrated that QT-RRE cells expressed higher phospho ERK1/2 levels than LLC-PK 1 cells ( Fig. 1 B, top ). Treatment of QT-RRE cells with PD-98059 decreased the levels of phospho ERK1/2. There was little change in total ERK1/2 or actin protein expression following treatment of cells with PD-98059 ( Fig. 1 B, middle and bottom ).
Fig. 1. ERK activity is elevated in tuberin-negative cells. Quinol-thioether-transformed rat renal epithelial (QT-RRE) cells were incubated with 50 µM PD-98059. Lysates were harvested to assay ERK 1/2 activity ( A ) and to determine phospho ERK1/2, ERK 1/2, and actin expression ( B ). For the ERK1/2 activity assay, ERK was immunoprecipitated from 500 µg of soluble protein using a goat anti-ERK1 polyclonal antibody (C-16, Santa Cruz Biotechnology) followed by protein G-Sepharose. The immunoprecipitates were washed extensively and assayed for kinase activity using MBP as a substrate. The incorporated [ - 32 P]ATP was quantified by liquid scintillation spectroscopy. LLC-PK 1 cells serve as negative control. Values represent means ± SE ( n = 3). ERK activity in QT-RRE was 0.15 ± 0.02 pmol pi·min -1 ·µg protein -1. A significant difference was seen between QT-RRE cells and treatment groups and LLC-PK 1 cells at * P < 0.01.
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.
Fig. 2. Elevated ERK activity in QT-RRE cells is dependent on protein tyrosine kinase activity. QT-RRE cells were treated with genistein (100 µM), an inhibitor of protein tyrosine kinases, for 1 h. Total protein was isolated from the cells and 2 µg of protein were used for the measurement of ERK activity. The incorporated [ - 33 P]ATP was quantified by liquid scintillation spectroscopy. Values represent means ± SE ( n = 3). A significant difference was seen between control and treatment groups at * P < 0.05 and ** P < 0.01.
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.
Fig. 3. Elevated ERK activity in QT-RRE cells is independent of the epidermal growth factor receptor (EGF-R). Cells were treated with AG-1478 (200 nM), a specific inhibitor of EGF-R, and/or EGF (20 nmol/ml) for 30 min. Total protein was isolated from the cells and 2 µg of protein were used for the measurement of ERK activity. The incorporated [ - 33 P]ATP was quantified by liquid scintillation spectroscopy. Values represent means ± SE ( n = 3). A significant difference was seen between control and treatment groups at * P < 0.01. NRK52-E cells served as a positive control for EGF-R activation.
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. 4 A, top ) had little effect on total ERK 1 and 2 expression in QT-RRE 1 and 2 ( Fig. 4 A, middle ). There was little change in actin expression ( Fig. 4 A, 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. 4 B ), which is consistent with the view that decreases in ERK activity subsequent to restoration of tuberin expression contribute to the suppression of cell proliferation.
Fig. 4. Tuberin suppresses 3 thymidine incorporation. Transient transfection with Tsc-2 cDNA was performed as described in MATERIALS AND METHODS and lysates were harvested for Western blot assay to measure changes in tuberin, ERK 1/2, and actin expression ( A ). The cells were incubated with 5 µCi/ml of 3 thymidine for 4 h. Incorporation of 3 thymidine into newly synthesized DNA was determined by liquid scintillation spectroscopy. Values are means ± SE ( n = 3; B ). A significant difference was seen between control and Tsc-2-transfected cells at * P < 0.05.
Downregulation of cyclin D 1 expression by tuberin. Cyclin D 1 is a key regulator of G 1 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 D 1 in QT-RRE cells by Western blot analysis. QT-RRE cells express high levels of cyclin D 1, 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 D 1. Expression of tuberin again had little effect on actin (housekeeping protein) expression in QT-RRE cells (data not shown).
Fig. 5. Restoration of tuberin suppresses cyclin D 1 levels. A : Western blot analysis was performed with protein extracts obtained from QT-RRE cells either transfected with vector alone (control) or Tsc-2 cDNA. Total protein (60 µg) was loaded onto 12% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to PVDF membranes and the membranes were incubated with anticyclin D 1 antibody (A-12, Santa Cruz Biotechnology) followed by anti-mouse secondary antibody. The proteins were visualized by the ECL system. B : autoradiogram was quantitated using chemimager and the intensity of each band was presented as a percentage of QT-RRE1.
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. 6 A ). B-Raf and Raf-1 activity were subsequently measured following immunoprecipitation. B-Raf activity was higher in QT-RRE cells compared with LLC-PK 1 cells, although activity varied within the three cell lines ( Fig. 6 B ). 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. 6 C ). Levels of Raf-1 in QT-RRE cells were also five- to ninefold higher than those in NRK-52E and LLC-PK 1 cells ( Fig. 7 A ). 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. 7 B ).
Fig. 6. Tuberin reduces B-Raf activity in QT-RRE cells. A : RT-PCR detection of B-Raf expression. B : B-Raf activity in QT-RRE cells. Data represent the average of 2 experiments. LLC-PK 1 cells serve as a negative control. C : restoration of tuberin suppresses B-Raf activity. Transient transfection of QT-RRE cells, RT-PCR of B-Raf and its activity were determined as described in MATERIALS AND METHODS. Values represent means ± SE ( n = 3). B-raf activity in QT-RRE was 0.15 ± 0.02 pmol pi·min -1 ·µg protein -1. A significant difference was seen between untransfected and Tsc-2 cDNA-transfected QT-RRE cells at * P < 0.01.
Fig. 7. Tuberin reduces Raf-1 activity in QT-RRE cells. A : Raf-1 activity in QT-RRE cells. B : restoration of tuberin suppresses Raf-1 activity. NRK-52E and LLC-PK 1 cells serve as negative controls. Transient transfection of QT-RRE cells, and Raf-1 activity were determined as described in MATERIALS AND METHODS. Raf-1 activity in nontransfected QT-RRE was 0.014 ± 0.002 pmol pi·min -1 ·µg protein -1. Values represent means ± SE ( n = 3). A significant difference was seen between negative controls and QT-RRE cells at * P < 0.01.
DISCUSSION
We used QT-RRE cell lines lacking tuberin expression to test the hypothesis that loss of tuberin function disrupts the ERK-signaling pathway. This was accomplished by measuring the activity of key components of the ERK cascade in QT-RRE cells and normal kidney epithelial cells and by examining the consequences of the restoration of tuberin into QT-RRE cells on the activity of these components. QT-RRE cells express significantly higher basal ERK activity than other renal epithelial cell lines, indicating that ERK is constitutively activated in these cells ( Fig. 1 ). The constitutive activation of the ERK-signaling pathway occurs in many types of human tumor cell lines and primary tumors, and overstimulation of the ERK pathway may play a role in tumorigenesis ( 5 ). Activated ERKs phosphorylate and activate a number of protein substrates, including other downstream kinases and transcription factors. Aberrant expression and/or activation of these proteins may predispose cells to undergo neoplastic transformation. Constitutive activation of the ERK pathway contributes to cell transformation ( 14, 22 ) and may similarly contribute to ROS-induced cell transformation during the genesis of QT-RRE cell lines.
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. 6 C ) 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. 6 A ).
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. 6 B and 7 A ). However, whereas restoration of tuberin in these cells remarkably decreased B-Raf activity ( Fig. 6 B ), Raf-1 activity was only slightly diminished ( Fig. 7 B ), 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 D 1 expression ( 39 ). This elevation is likely related to high ERK activity in QT-RRE cells ( Fig. 1 ). Restoration of tuberin expression decreased cyclin D 1 expression in QT-RRE cells ( Fig. 5 ), consistent with the previous finding that Tsc-2 antisense oligonucleotides increase cyclin D 1 expression ( 25 ). Cyclin D 1 is a downstream effector of ERK ( 12 ), although the molecular events leading to increases in cyclin D 1 expression subsequent to ERK activation remain unclear. Our results provide strong evidence coupling tuberin to cell cycle progression. As predicted by cyclin D 1 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 D 1 ) ( 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 ).
Fig. 8. Putative mechanisms for the effects of tuberin on MAP kinase pathway signaling. Tuberin exhibits GTPase-activating protein (GAP) activity toward Rap1 ( 35 ), and our data show that restoration of tuberin decreased B-Raf and ERK1/2 activity in QT-RRE cells. The decrease in ERK1/2 activity is likely mediated via a decrease in MEK activity. The reduction in ERK1/2 by tuberin could also mediate decreased cyclin D 1 expression and the decreased rate of new DNA synthesis in tuberin-expressing QT-RRE cells.
In summary, we have shown that restoration of tuberin in QT-RRE cells decreases ERK and B-Raf activity and the expression of cyclin D 1. 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 D 1 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.
GRANTS
This work was supported by Grant GM-39338 to S. S. Lau. We also acknowledge the support of National Institute of Environmental Health Sciences (NIEHS) Center Grant ES-07784 and NIEHS Training Grant ES-07247 to M. A. S. Chacko.
ACKNOWLEDGMENTS
The authors thank Dr. R. S. Yeung at the University of Washington, Seattle, WA, for kindly providing Tsc-2 cDNA used in these studies.
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作者单位：Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Texas 78712