Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR (original) (raw)
Tsc2-null fibroblasts display early senescence. To investigate the growth characteristics of cells lacking Tsc2, we prepared cultures of early-stage mouse embryos derived from Tsc2+/– interbreedings. MEF cultures were established by standard methods from viable E10.5–11.5 _Tsc2–/–_embryos and their littermates. Tsc2–/– MEF cultures uniformly displayed more limited growth in culture than Tsc2+/– or Tsc2+/+ MEFs, failing to divide after P8 (Figure 1a). In contrast, Tsc2+/– and Tsc2+/+ MEFs continued to expand past P10 in all cases, with growth plateauing after about ten divisions in most cases. Both types of cell lines gave rise spontaneously to immortalized, rapidly growing derivative cell lines between P10 and P25 (Figure 1a). For the Tsc2+/– and Tsc2+/+ cultures, this occurred in all cases (n = 5). For Tsc2–/– cultures, however, this occurred in one of five cases.
Premature senescence of Tsc2–/– MEF cultures. (a) Growth curves of cultured Tsc2–/– (open squares), Tsc2+/– (filled circles), and wild-type (filled triangles) MEFs. PDL, population doubling. (b) Phase-contrast view of senescent P8 Tsc2–/– (left) and P8 control (right) MEFs. (c) Immunoblot analysis of extracts from primary MEF cultures of the indicated genotypes. Note the increased expression of p21CIP1/WAF1 by the Tsc2–/– MEFs. (d) Immunoblot analysis similar to that shown in c of TP53–/– MEF lines with various Tsc2 genotypes. (e) Left, immunoblot analysis of tuberin, hamartin, and ERK expression by MEFs with various genotypes. Note reduced expression of tuberin in the Tsc1-null MEFs. Right, tuberin immunoprecipitation (IP) from starved and stimulated (30 minutes) TP53–/– cells shows no change in tuberin or hamartin expression levels or association.
Phase-contrast views of Tsc2–/– cells at P8 to P12 demonstrate that they have features that are indicative of senescence (Figure 1b). Cells are large with relatively large processes, and they fail to incorporate BrdU, indicating cell-cycle arrest. FACS analysis of cell-cycle distribution at P10 showed that they had normal DNA content and were 81% G1 and 19% G2 plus M phase, compared with 49% G1, 3% S, and 48% G2 plus M distribution for Tsc2+/+ MEFs at identical passage. Immunoblot analysis of cell lysates demonstrated that the Tsc2–/– MEFs had elevated levels of the cdk inhibitor p21CIP1/WAF1 compared with Tsc2+/– and Tsc2+/+ MEFs of identical passage (Figure 1c). In contrast, levels of the p27KIP1 cdk inhibitor, previously implicated in growth-signaling abnormalities in Tsc2-null rat embryo fibroblasts (19), as well as the cdk inhibitor p16INK4a, were no different among the different types of MEFs.
Rescue of Tsc2–/–-induced senescence by p53 loss. To circumvent the premature senescence of Tsc2–/– MEFS, mice bearing the Tsc2– allele were interbred with mice carrying a null allele for TP53 to obtain TP53–/–Tsc2+/– mice. Breeding studies showed that being null for TP53 did not improve the survival of Tsc2–/– embryos, because none survived past E12.5 and double-null embryos at E10.5 often were more poorly developed than littermates that were also TP53–/– but Tsc2+/– or Tsc2+/+. Viable E10.5 TP53–/–Tsc2–/– embryos gave rise to MEF cultures that grew rapidly and indefinitely without senescence (five of five embryos), similar to TP53–/–Tsc2+/– (four of four) and TP53–/–Tsc2+/+ (three of three) cultures derived from littermates.
There were no significant differences in growth rates in 10% serum among these cell lines (see below; Figure 3b, top). In short-term growth experiments in the absence of serum, however, TP53–/–Tsc2–/– MEFs displayed continuing growth in contrast to control TP53–/– MEFs (Figure 3b, bottom). The p21CIP1/WAF1 levels were undetectable in extracts from these MEFs (data not shown), while levels of p27KIP1 and p16INK4a were equivalent (Figure 1d).
Effects of inhibitors on S6K/S6 phosphorylation and growth of TP53–/–Tsc2–/– and control TP53–/– MEFs. (a) Immunoblot analysis of a serum-starved (2 days) then stimulated TP53–/–Tsc2+/+ cell line (top) and a serum-starved TP53–/–Tsc2–/– cell line (bottom). Cells were treated with 10 μM LY294002, 0.1 μM wortmannin, 10 nM rapamycin, 0.1 μM calyculin A, 5 μM TPCK, 10 μM U0126, or 20 μM PD98059 for 30 minutes. (b) Growth effects in two TP53–/–Tsc2–/– (open squares) and two TP53–/–Tsc2+/+ (filled triangles) cell lines of treatment with rapamycin. Each symbol reflects a consecutive day in culture. Rapamycin selectively reduces the growth of the TP53–/–Tsc2–/– cell lines in 0% serum. (c) Immunoblot analysis of effects of 1- and 2-butanol treatment on S6K/S6 phosphorylation in TP53–/–Tsc2–/– and control TP53–/– cell lines. 1- or 2-butanol (0.3%) were applied to the cell lines for 30 minutes, and the cells were serum stimulated for 5 minutes. (d) Immunoblot analysis of effects of AA deprivation and stimulation on S6K/S6 phosphorylation in two TP53–/–Tsc2–/– cell lines. All treatments were for 2 hours. Note that with either the EBSS or HBSS buffers, AAs are required to maintain pS6K and pS6 phosphorylation.
Expression of tuberin and hamartin was examined in these TP53–/–Tsc2–/– cell lines and compared with Tsc1–/– cell lines described previously (5). Tuberin levels were reduced by over half in Tsc1–/– cell lines compared with controls, while hamartin levels were not affected by loss of tuberin (Figure 1e). In addition, there was no change in tuberin or hamartin levels, or their physical association in control MEF cells in response to PDGF or serum stimulation (Figure 1f).
Growth-signaling alterations in Tsc2–/–TP53–/– cells. The growth advantage that we detected in the absence of serum suggested that growth stimulatory pathways might be constitutively active in Tsc2-null cells. This idea was supported by our previous studies indicating that 4E-BP1, S6K, and S6 were constitutively phosphorylated and activated in Tsc1-null cells (5). Therefore, we examined several signaling pathways relevant for control of cell growth, using the TP53–/– MEF lines in serum-starved cultures with or without serum refeeding (Figure 2a). Expression of ERK isoforms was similar in these MEFs irrespective of genotype. In comparison with control cells, two TP53–/–Tsc2–/– MEF lines showed a reduction in the level of activated ERK1 and ERK2 in response to serum stimulation (Figure 2a, first and seventh pair of lanes), but this was not seen in all TP53–/–Tsc2–/– MEFs (Figure 2a, third and fourth pair of lanes). Activation of p90RSK by serum was similar in all of these cell lines. In contrast, activation of Akt in response to serum was markedly reduced in all of the TP53–/–Tsc2–/– cells, as assessed by phosphorylation at Ser473. Moreover, S6K was constitutively activated in all of the Tsc2-null cells in the absence of serum, as assessed by phosphorylation at Thr389, and showed only a modest increase in phosphorylation with serum stimulation (Figure 2a). Consistent with constitutive activation of S6K, pS6 levels (Ser235/236) were also increased in the TP53–/–Tsc2–/– cells in the absence of serum and did not increase with serum refeeding (Figure 2a). In these experiments we examined several TP53–/–Tsc2+/+ and TP53–/–Tsc2+/– MEF lines and saw no difference between them, indicating that haploinsufficiency for Tsc2 had no effect on these pathways (Figure 2a).
Growth signaling in TP53–/–Tsc2–/– MEFs and Tsc2+/– mouse cystadenomas. (a) Immunoblot analysis in serum-starved (2 days) or serum-fed (30 minutes) TP53–/– MEFs. Genotypes are indicated across the top. Note that tuberin is not expressed in the Tsc2–/– cell lines; pAkt levels increase very little in the Tsc2–/– cell lines in response to serum, and pS6K and pS6 levels are increased in the Tsc2–/– cell lines without serum addition. (b) Immunoblot analysis on renal cystadenomas derived from Tsc2+/– mice. Note the presence of pS6 in all three cystadenomas. (c) Immunoblot analysis of a revertant TP53–/–Tsc2–/– cell line expressing TSC2. Note the decrease in pS6K and pS6 levels in the revertant line during serum starvation. pS6 S235/236, pS6 (Ser235/236).
We also examined the state of activation of this pathway in tumors derived from Tsc2+/– mice, which are known to show loss of heterozygosity for the wild-type Tsc2 allele in most cases (4). ERK and eIF4E levels were similar, while pS6 was present only in cystadenomas and not in control kidney tissue of these mice (Figure 2b). We also generated a revertant, TSC2-expressing stably transfected cell line from a TP53–/–Tsc2–/– cell line. In the revertant cell line, expression of pS6K and pS6 under conditions of serum starvation was markedly reduced, similar to TP53–/– control cell lines (Figure 2c).
In summary, we have shown that there is constitutive high-level activation of S6K in the Tsc2-null TP53–/– cell lines as well as Tsc2-null tumors from Tsc2+/– mice. In contrast, Akt activation is markedly reduced in response to growth factor stimulation in Tsc2-null lines. These findings were entirely similar in both early-passage (P5) and late-passage (greater than P20) Tsc2-null TP53–/– cell lines (data not shown).
Inhibitors of mTOR specifically revert the S6K activation and growth phenotype of TP53–/–Tsc2–/– cells. These findings, as well as previous observations (5, 11, 20), implicated an abnormality in signaling at approximately the level of mTOR in cells lacking Tsc2. To explore this defect, we examined the effects of treatment with several inhibitors of the kinases in this pathway (Figure 3a). In serum-starved TP53–/–Tsc2–/– cells, treatment with either 10 nM rapamycin or 10 μM LY294002 abolished the phosphorylation of S6, consistent with their actions in inhibiting mTOR (21, 22). Wortmannin (0.1 μM) (inhibits PI3K; ref. 23), however, had no effect on phosphorylation of S6 in the serum-starved TP53–/–Tsc2–/– cells. Treatment of the Tsc2-null cells with three additional inhibitors, TPCK (inhibits PDK1; ref. 24), U0126 (inhibits MEK kinase; ref. 25), and PD98059 (inhibits MAPKK; ref. 26), also failed to have significant effects on the levels of pS6, while calyculin A (serine-threonine phosphatase inhibitor; ref. 27) slightly increased S6 phosphorylation (Figure 3a). In aggregate, these observations suggest that the activation of S6K in Tsc2-null cells is dependent upon functional mTOR but independent of PI3K, PDK1, or MEK kinases (22–27). Analysis of control serum-stimulated TP53–/– lines demonstrated that these compounds all had their predicted effects (Figure 3a, top).
We also assessed the importance of functional mTOR on the growth characteristics of the TP53–/–Tsc2–/– cells. The persistent growth of the Tsc2-null cells in the absence of serum was reduced by rapamycin treatment in the range of 50 pM to 50 nM (Figure 3b). Even in 50 nM rapamycin, however, the growth behavior of the TP53–/–Tsc2–/– lines was still distinct from that of Tsc2 wild-type TP53–/– lines, perhaps reflecting short-term persistence of a growth advantage despite effective mTOR inhibition. Analysis of cell extracts from these cells under these conditions confirmed that the activation of S6K and S6 was abolished by rapamycin at doses from 50 pM to 50 nM (data not shown).
To explore the importance of other stimuli in the regulation of mTOR activity in Tsc2-null cells, we examined the effects of reduction in phosphatidic acid (PA) and AA levels (28, 29). Treatment with 1-butanol or 2-butanol to reduce cellular PA levels resulted in a reduction in pS6K levels in both serum-starved and serum-stimulated TP53–/–Tsc2–/– cells and had similar effects on serum-stimulated control TP53–/– cells (28) (Figure 3c). Exposure of previously serum-starved TP53–/–Tsc2–/– cell lines to an absence of AA led to a significant reduction in pS6K and pS6 levels, while addition of fresh AA appeared to increase pS6K and pS6 levels to a small extent (Figure 3d).
Analysis of the tuberin-hamartin complex and potential interactors. Since these observations suggested a potential interaction between Tsc1/Tsc2 and mTOR, we performed an in vitro kinase assay combining immunopurified mTOR, immunopurified Tsc1/Tsc2, γ32P-ATP, and bacterially expressed 4E-BP1 (Figure 4a). Phosphorylation of both mTOR (autokinase activity) and 4E-BP1 were seen under these conditions, but was not influenced by incubation with Tsc1/Tsc2. We also assessed the possibility that Tsc1/Tsc2 might influence phosphatase activity on mTOR kinase substrates. Labeled 32P-4E-BP1 was added to cell extracts from control or TP53–/–Tsc2–/– cells, and there was no difference in phosphatase activity among the different samples (Figure 4b).
The mTOR functional analysis and purification of a Tsc1/Tsc2 complex. (a) Autoradiograph from an mTOR kinase reaction. Addition of mTOR and Tsc1/Tsc2 are indicated at the top. Equivalent amounts of mTOR autokinase activity and kinase activity on 4E-BP1 are seen whenever mTOR is included. C, anti-C20 tuberin Ab; N, anti-N19 tuberin Ab; E, eluate. (b) Autoradiograph of a phosphatase assay. 4E-BP1 was phosphorylated in vitro using γ32P-ATP and then was included as a substrate to assess phosphatase activity of two TP53–/–Tsc2–/– and two TP53–/– control cell line extracts. There is no difference in the level of phosphatase activity. (c) Coomassie blue–stained gel showing successive steps in the purification of TSC1/TSC2 from brain extracts. Material bound to an anti-TSC1 affinity (H2 Ab) column, residual on the column after elution with peptide, the eluate, and the material obtained from an anti-TSC2 (C20) Ab column are shown in successive lanes. The location of 14-3-3γ is indicated by an asterisk. (d) Immunoblot analysis of Tsc1/Tsc2–binding partners. IP was performed with the indicated Ab’s (Tsc2 N19, C20) followed by immunoblotting. Tsc2 row: + indicates extract from a control TP53–/– cell line; – indicates a Tsc2–/–TP53–/– cell line.
To identify potential interactor(s) of Tsc1/Tsc2, we directly purified the Tsc1/Tsc2 complex from mouse brain by serial immunoaffinity chromatography (Figure 4c). A complex consisting of Tsc2 (tuberin) and Tsc1 (hamartin) could be purified (confirmed by mass spectrometry), demonstrating the highly stable binding between these proteins at approximately 1:1 stoichiometry. One band of size 30 kDa was identified as being 14-3-3γ. This was confirmed by immunoblotting (Figure 4d). Several other fainter bands were seen in the purified material, all present at a stoichiometry of much less than 1:1. Akt and mTOR, previously described (11, 13) as occurring in association with Tsc1/Tsc2, were not present in this purified complex (Figure 4d).
Reduction in PI3K-Akt signaling in TP53–/–Tsc2–/– and Tsc1–/– cells. To understand the major reduction in serum-stimulated activation of Akt in Tsc2-null cells (Figure 2a), we explored whether there was a change in kinetics rather than the extent of Akt activation. Activation of Akt (assessed by phosphorylation at Ser473) was markedly reduced in the Tsc2–/–TP53–/– cells at all time points (10, 30, 60, and 90 minutes) in comparison with control TP53–/– cells, and the kinetics were no different from control cells (Figure 5a). A marked difference in the level of Akt activation in Tsc1- and Tsc2-null cells compared with control cells persisted after treatment with calyculin A (Figure 5b), suggesting that the difference was due to a lack of phosphorylation of Akt in both Tsc1- and Tsc2-null cells rather than differences in phosphatase activity. To explore this further, we assessed PI3K activity in these cells. Using both serum at 10% (data not shown) and PDGF at 50 ng/ml stimulation, there was a significant reduction in the amount of 3′-phosphoinositides generated by phosphotyrosine or PDGFRβ immunoprecipitates from the Tsc2–/–TP53–/– cells compared with TP53–/– control cells (Figure 5c).
Reduced PI3K-Akt signaling in Tsc2–/–TP53–/– and Tsc2–/– cells. (a) Immunoblot analysis of cell extracts showing reduced pAkt in Tsc2–/–TP53–/– in comparison to control TP53–/– cells at all time points after 10% serum addition. pAkt S473, pAkt (Ser473). (b) Immunoblot analysis showing reduced pAKT in serum-starved Tsc1–/– and Tsc2–/–TP53–/– cells with and without treatment with calyculin A. (c) Autoradiography demonstrates reduced PI3K activity in Tsc2–/–TP53–/– cells in comparison with TP53–/– cells, in response to PDGF. p-tyrosine, phosphotyrosine; PIP, phosphoinositide phosphate. (d) Ruffling activity, indicated by arrowheads, in response to PDGF was reduced in Tsc2–/–TP53–/– cells in comparison to control TP53–/– cells or a revertant TSC2-expressing cell line. Error bars (n = 3) depict the SD. (e) YPH-Akt translocation is reduced in Tsc2–/–TP53–/– cells in comparison to control TP53–/– cells or a revertant TSC2-expressing cell line. PDGF stimulation leads to uniform YFP staining of the plasma membrane in Tsc2+/+ and revertant cells, but not in Tsc2–/– cells. The staining intensity in cross-sections of the same cells is shown in the graphs at right. AU, arbitrary units.
As a measure of the response to PDGF stimulation, we also examined the degree of ruffling and of intracellular translocation of a reporter molecule for D3-polyphosphoinositide generation, consisting of the PH domain of Akt fused to YFP (YPH-Akt). The Tsc2–/–TP53–/– cells displayed reduced ruffling activity in response to stimulation with PDGF compared with control cell lines (Figure 5d). In addition, there was a marked reduction in the extent of translocation of the YPH-Akt reporter protein (Figure 5e). In control cells, translocation of the reporter to the cell surface in response to PDGF treatment resulted in a uniform staining appearance, while in the Tsc2–/–TP53–/– cells the YPH-Akt showed a generalized cytoplasmic location that did not change with PDGF treatment. The revertant cell line in which TSC2 expression was restored had a ruffling response and YPH-Akt translocation that was similar to the control Tsc2-expressing TP53–/– cell lines (Figure 5, d and e).
These results demonstrated that there was a major reduction in PI3K activity and Akt activation in cells lacking Tsc2 in response to both serum and PDGF. We then examined the possibility that differences in receptor amount or activation resulted in these differences in PI3K activation. Expression of PDGFRβ and PDGFRα was markedly reduced in the TP53–/–Tsc2–/– as well as Tsc1–/– cells in comparison to control cell lines (Figure 6a). In contrast, there was little or no difference in insulin receptor expression in these cell lines (Figure 6a). In addition, reduction in PDGFRβ expression was seen in renal cystadenomas from the Tsc1+/– and Tsc2+/– mice in comparison with normal renal tissue (Figure 6a). Furthermore, the extent of phosphorylation of PDGFRβ in response to PDGF treatment was greatly reduced in Tsc2-null cell lines (Figure 6b). The amount of PI3K p85 subunit bound to the PDGFRβ receptor was also concordantly reduced, explaining the lack of an increase in 3′-phosphoinositide synthesis in Tsc2-null cells. In the revertant cell line in which TSC2 expression was restored, pAkt generation in response to PDGF treatment was normal (Figure 6b, right). Expression of the EGFR and the p85 subunit of PI3K showed little or no difference in the TP53–/–Tsc2–/– compared with the TP53–/– cell lines (Figure 6b). The majority of these studies were performed on the TP53–/– cell lines of greater than P20. Observations on expression of PDGFRβ and PDGFRα, however, and stimulation of Akt were entirely similar in these cell lines at P5 (data not shown), however.
PDGFR is reduced in Tsc2–/–TP53–/– and Tsc1–/– cells. (a) Immunoblot analysis of cell extracts demonstrates that both PDGFRβ and PDGFRα levels are reduced in both Tsc1-null and Tsc2-null cell lines compared with controls, while expression of insulin receptor α (IRα) is similar in these cells. Expression of PDGFRβ is reduced in tumor (T) extracts compared with normal kidney (NK) from both Tsc2+/– and Tsc1+/– mice. (b) Left top five rows, immunoblot analysis of cell extracts showing reduced amount of PDGFRβ in the Tsc2–/–TP53–/– cell line compared with the TP53–/– control. Left bottom three rows, analysis of PDGFRβ immunoprecipitations (IPs) showing reduced amount of PDGFRβ, pPDGFRβ, and bound PI3K p85 subunit in the Tsc2–/–TP53–/– cell extracts. Right, immunoblot showing expression of PDGFRβ is restored in a TSC2-expressing revertant TP53–/– cell line (pEF6/TSC2). (c) Autoradiogram showing levels of PDGFRβ in a pulse-chase experiment with S35-methionine labeling in TP53–/– cells. Levels of PDGFRβ are decreased at the 0 time point in both of the Tsc2–/– cell lines compared with controls, but levels decline similarly during the chase. (d) BrdU incorporation experiment for serum-starved Tsc2-null, control, and revertant TP53–/– cells in response to treatment for 12 hours with 25 ng/ml PDGF with or without 25 μM AG17 or 50 ng/ml EGF and 10 μM BrdU. Left, serum starvation for 1 day; right, for 3 days. Similar results were obtained using cell counting.
Metabolic labeling studies performed on TP53–/–Tsc2–/– and control TP53–/– lines demonstrated that there was reduced production of the PDGFRβ in the null cell lines, but that the half-life of PDGFRβ was similar in the two types of cells (Figure 6c). Additional evidence for the reduced function of PDGFR in cells lacking Tsc2 was the minimal growth effects of PDGF treatment with or without AG17 (PDGFR inhibitor; ref. 30) in TP53–/–Tsc2–/– cells (Figure 6d). In contrast, the control TP53–/– and the revertant cell lines showed the expected growth response to PDGF treatment that was inhibited by AG17. In addition, the EGF growth response was similar in both TP53–/–Tsc2–/– and control TP53–/– cells, consistent with the normal levels of EGFR expression by both cell types (Figure 6d).
To confirm that the lack of PDGFR expression was the cause of reduced activation of Akt in Tsc2-null cells, we used ectopic expression of PDGFRβ. TP53–/–Tsc2–/– cells expressing the PDGFRβ and YPH-Akt by transient transfection demonstrated normal translocation of the reporter protein to the cell surface in response to PDGF treatment, with a uniform staining appearance (Figure 7a). Moreover, TP53–/–Tsc2–/– cells transfected to express PDGFRβ demonstrated enhanced activation of Akt in response to PDGF, as well as EGF, serum, and insulin (Figure 7b).
Correction of Akt signaling in Tsc2–/–TP53–/– cells by PDGFRβ transfection. (a) YPH-Akt translocation. Tsc2–/–TP53–/– cells were cotransfected with YPH-Akt and PDGFRβ or an empty vector and were serum starved overnight before PDGF addition. The left and right panels are fluorescent images of the cells before and 4 minutes after PDGF addition, respectively. PDGF stimulation leads to uniform YFP staining of the plasma membrane in cells transfected with the PDGFRβ, but not in cells transfected with empty vector. The staining intensity in cross-sections of the same cells is shown in the graphs at right. (b) Recovery of Akt activation. Immunoblot analysis of Tsc2–/–TP53–/– cells transfected with empty vector (left) or PDGFRβ (right) that were serum starved and then stimulated with 50 ng/ml PDGF-BB (P), 100 ng/ml EGF (E), 10% serum (S), or 0.1 μM insulin (I). Note the increase in the Akt phosphorylation in the PDGFR-transfected cells in response to all stimuli.