Hypoxia Causes Inhibition of Protein Translation in S2 Cells
Hypoxia down-regulates protein synthesis in Drosophila embryos and in mammalian tissue culture cells. We examined the effects of hypoxia on translation by assaying [35S]-methionine incorporation by Drosophila S2 cells in culture. Different levels of hypoxia were achieved by admixing an inert gas (nitrogen or argon) and air, and incorporation was measured between the first and second hour after hypoxia exposure. Radiolabel incorporation declined with increasing hypoxia, reaching ~75% inhibition at 0.5% oxygen (A). To assess the speed of onset, we measured [35S]-methionine incorporated during 1-h intervals at different times after exposure of cells to 0.5% oxygen. There was an immediate drop in incorporation (~60% inhibition) at 1 h followed by continued, slower decline so that at 16 h, there was ~85% inhibition (B). No decline in cell viability occurred during overnight hypoxia (<0.1% O2; A). Our results show that, like other cells that have been examined, Drosophila S2 cells exhibit a rapid and dramatic suppression of translation in response to hypoxia.
Figure 1. Hypoxia induces inhibition of protein translation in Drosophila S2 cells. (A) Protein translation is inhibited in an oxygen concentration-dependent manner. Cells were exposed to the indicated concentration of O2 for 1 h, [35S]-methionine was then added, (more ...)
Figure 6. Lack of Ptp61F improves cell survival under hypoxia. (A) Cells deficient in Ptp61F displayed enhanced survival under hypoxia compared with control cells (LacI, GFP, or luciferase RNAi). *p < 0.05. (B and C) Inhibition of TORC1 activity abolishes (more ...)
To assess whether hypoxia would block induction of new gene expression, we examined a stable cell line harboring a GFP-tagged transgene expressed under the Cu2+-inducible methallothioneine promoter (A). Overnight exposure to CuSO4 induced bright fluorescence in normoxic cells but not hypoxic cells (~0.5% O2). This hypoxia suppression of GFP expression was readily detected by fluorescence microscopy and by Western blot analysis (B and Supplemental Figure S1). In contrast to the protein, induction of GFP mRNA was not reduced by hypoxia (E), indicating that the inhibition of GFP expression is mainly due to suppression of translation.
Figure 2. Genome wide RNAi screening in S2 cells identifies sets of genes required for efficient suppression of translation in response to hypoxia. (A) Cu2+-inducible GFP reporter. (B) The expression of a GFP reporter gene is inhibited under hypoxia. CuSO4 (400 (more ...)
Genome-wide RNAi Screening Identifies Genes Involved in Inhibition of GFP Induction under Hypoxia
To probe the relevant mechanisms of oxygen sensing and the signaling steps leading to suppression of gene expression, we used a library of 7216 dsRNAs, representing the conserved genes of Drosophila
(Foley and O'Farrell, 2004
), in an RNAi screen for genes required for suppression of GFP induction in hypoxia. Our starting premise was that the suppression of new gene expression by hypoxia was not merely a passive consequence of cellular energy depletion but represented an active response requiring specific effector functions.
We added dsRNA to S2 cells harboring the GFP reporter and incubated these for 4 d. After 4 d of dsRNA treatment, CuSO4 was added and the cells were placed under hypoxia (~0.5% O2) overnight (C). After this period of induction in hypoxia, cells were fixed and screened for a GFP signal. In the first round of screening, we visually identified wells where the dsRNA allowed substantial GFP induction. We rescreened candidates by the same test, and then assessed the degree of the bypass of the hypoxia-induced inhibition by Western blotting for GFP (D).
The genes identified in our screen are summarized in . We identified sets of genes involved in signaling pathways, including Tsc1, Tsc2, Ptp61F, SH3PX1, and MESR4. Among them, Tsc1 and Tsc2 form a complex (TSC complex), and have been reported previously to be involved in inhibition of protein translation under hypoxia. We also recovered several genes involved in mitochondrial protein transport (Tom40, Tim44) as well as in mitochondrial function (CG4589, CG10691). Some of the candidates are components of translation machinery (eIF2Bβ; D) or are known to negatively regulate protein translation (Musashi). We discuss the screen further below, but focus results in this report on the TOR pathway and involvement of Ptp61F in the regulation of translation in hypoxia.
Genes required for hypoxia-induced inhibition of protein translation in Drosophila S2 cells
TSC Complex and Ptp61F Are Required for Efficient Inhibition of Protein Translation under Hypoxia
Consistent with the involvement of TOR in promoting protein translation in other contexts (Ellisen, 2005
), addition of the TOR inhibitor rapamycin to normoxic S2 cells suppressed GFP induction as assessed by Western blots at 16 h after induction (A, lane 2 vs. lane 4). The identification of Tsc1 and Tsc2 in our screen suggests that down-regulation of TOR contributes to suppression of gene expression during hypoxia. Phosphatase and tensin homologue (PTEN), another negative regulator in the TOR pathway, was not recovered in our screens. However, a direct test of PTEN RNAi revealed that it results in cell death in Drosophila
S2 cells, which precluded test of a contribution to the response to hypoxia. In contrast to the dramatically increased GFP accumulation induced during hypoxia after RNAi of Tsc1 or Tsc2 (A, lane 3 vs. lanes 6 and 7), HIF-1 knockdown had no detectable effect (A, lane 3 vs. lane 5). These results indicate that down-regulation of TOR plays an important role in robust suppression of GFP expression induced under hypoxia and that this regulation is independent of HIF-1.
Figure 3. Ptp61F is required for efficient inhibition of protein translation under hypoxia. (A) The GFP reporter is induced in cells treated with RNAi against Tsc1, Tsc2, or Ptp61F. Experiments were performed as described in D. Rapamycin was added to 50 (more ...)
We hypothesized that, in addition to Tsc1 and Tsc2, our screen might identify other negative regulators operating at different points in the signal transduction pathway controlling TOR. One of the genes identified in our screen was of particular interest in this regard. Protein tyrosine phosphatase 61F (Ptp61F) has two homologues in mammals, protein tyrosine phosphatase 1B (PTP-1B) and the related protein TC-PTP. PTP-1B is localized in the membrane and negatively regulates the insulin receptor signaling and leptin signaling in mammals (Seely et al., 1996
; Goldstein et al., 1998
; Haj et al., 2003
). TC-PTP is localized in the nucleus, has a negative input on Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in mammals, and is important in immune homeostasis (Simoncic et al., 2002
Ptp61F has four different splice forms (A, B, C, and D); A and D are bound to membrane and B and C are localized in the nucleus (McLaughlin and Dixon, 1993
). Ptp61F interacts with dreadlocks
), which has been shown to bind to the insulin receptor (Song et al., 2003
), and it was recently reported to be a negative regulator of JAK/STAT pathways (Baeg et al., 2005
; Muller et al., 2005
). Although its roles in hypoxia have not been reported, we were attracted by the involvement of Ptp61F in pathways of potential relevance: insulin is an upstream regulator of the TOR pathway, and JAK/STAT has been implicated in stress responses.
The dsRNA used in most of our experiments targets all four splice forms. The effectiveness of RNAi knockdown of Ptp61F was confirmed by RT-PCR, and specificity was confirmed by knockdown by using an independent dsRNA targeting nonoverlapping sequences in Ptp61F (B) as well as by knockdown of specific isoforms (below). The magnitude of the Ptp61F contribution to hypoxia suppression of induced GFP expression was assessed by Western blot. As seen in A, Ptp61F-RNAi increased the level of induced GFP expression in hypoxic cells (A, lane 3 vs. lane 8), but to a smaller degree than the knockdown of Tsc1 or Tsc2 (A, lanes 6 and 7).
We examined [35S]-methionine incorporation as a measure of effects on translation (C). Cells were treated with dsRNA against Tsc2, Ptp61F or control RNAi for 4 d, and then they were exposed to normoxia or hypoxia treatment (<0.5% O2) for 16 h before addition of [35S]-methionine. Although Tsc2 knockdown seemed to cause a slight increase in [35S]-methionine incorporation during normoxia, this effect was not significant. Ptp61F had no evident effect on translation in normoxia. Hypoxia caused a clear suppression of incorporation as described above. Cells treated with dsRNA against Tsc2 or Ptp61F had a significantly higher level of incorporation than cells treated with control dsRNA under hypoxia (1.4–2.5-fold increase; p < 0.05). These results indicate that Ptp61F is required for efficient inhibition of protein translation under hypoxia.
Down-Regulation of TOR Activity under Hypoxia Requires Ptp61F
Because mammalian (m)TOR down-regulation contributes to the suppression of translation in mammalian cells, we tested TSC and Ptp61F contributions to down-regulation of TOR during hypoxia in Drosophila S2 cells. We measured TOR activity by examining phosphorylation of two well-characterized TOR substrates, p70S6K and 4E-BP (A). A modest level of p70S6K and 4E-BP phosphorylation was observed in control RNAi-treated cells in normoxia, and this phosphorylation was reduced in hypoxia, indicating that TOR activity declined in hypoxia (A, lane 1 vs. lane 2). Tsc2 RNAi dramatically increased TOR phosphorylation of 4E-BP in normoxia. This finding suggests that the TSC is active under normal growth conditions and that knockdown of its function results in supraphysiological levels of TOR activity. Tsc2-depleted cells sustained TOR activity under hypoxia, albeit with a slight decrease compared with normoxia (A, lanes 1 and 2 vs. lanes 7 and 8). Similarly, Ptp61F-depleted cells sustained TOR activity under hypoxia (A, lane 2 vs. lanes 4 and 6); however, Ptp61F RNAi had only a slight effect, if any, on the phosphorylation levels of p70S6K and 4E-BP in normoxia (A, lane 1 vs. lanes 3 and 5).
Figure 4. Ptp61F contributes to inhibition of protein translation under hypoxia by down-regulating TOR activity. (A) Ptp61F is required for down-regulation of TOR activity. Cells were treated with dsRNA against Tsc2 or Ptp61F, exposed to hypoxia (0.5% O2, 16 h), (more ...)
It has been reported that RNAi using long dsRNA may have off-target effects in Drosophila S2 cells. The selectivity of dsRNA against Ptp61F was demonstrated by rescuing the effects of RNAi by selective expression of Ptp61F. Using a dsRNA complementary to the 3′ untranslated region (UTR) of transcripts encoding Ptp61F membrane isoforms A and D, we were able to selectively knockdown the cognate transcripts (Supplemental Figure S3). Although we were unable to design UTR targeted dsRNAs capable of knocking out isoforms B and C, treatment with the dsRNA specific for the A and D isoforms was sufficient to cause an increased TOR activity during hypoxia (B, lanes 5 and 6, and Supplemental Figure S3). Expression of a cDNA that encodes the Ptp61F-A isoform but that lacks the 3′ untranslated region of the endogenous transcript restores Ptp61F-A to Ptp61F-A/D–depleted cells and also restored down-regulation of TOR activity under hypoxia (B, lanes 7 and 8). This shows that the effect we observed in cells treated with Ptp61F RNAi is indeed due to Ptp61F depletion rather than an off-target effect, and that Ptp61F functions as a negative regulator of the TOR pathway in hypoxia. Furthermore, these findings show that nonnuclear forms of Ptp61F play an important role in hypoxia down-regulation of TOR activity in Drosophila S2 cells.
Our findings suggest that TOR activity is down-regulated during hypoxia and that this down-regulation makes a substantial contribution to the suppression of translation in hypoxia. To determine whether any consequential residue of TOR function persists during hypoxia, we examined the effect of rapamycin on [35S]-methionine incorporation after 16 h of hypoxia. Rapamycin addition abolished phosphorylation of p70S6K in Drosophila S2 cells (Supplemental Figure S2A) and suppresses [35S]-methionine incorporation in normoxia, but it had little or no effect in hypoxia (C, control RNAi). This suggests that TOR activity is effectively suppressed during hypoxia, at least in so far as hypoxia eliminates the contribution of TOR activity to [35S]-methionine incorporation.
Knockdown of Ptp61F had little effect on [35S]-methionine incorporation in normoxia or its suppression by rapamycin (C), suggesting that Ptp61F did not substantially alter TOR contributions to translation in normoxia. When Ptp61F-knockdown cells were subjected to hypoxia, rapamycin suppressed the sustained level of [35S]-methionine incorporation to the levels seen in control cells in hypoxia (C, bottom). This result shows that knockdown of Ptp61F results in a sustained level of TOR during hypoxia, which promotes the sustained translation.
In a parallel set of experiments, Raptor RNAi was used to inactivate TOR, and its consequence on the ability to induce GFP was examined under normoxia and hypoxia. Raptor forms a complex with TOR and is required for TOR function as a positive regulator of protein translation. Raptor RNAi diminished the ability of Ptp61F RNAi to permit induction of GFP expression during hypoxia (Supplemental Figure S1). These results show that Ptp61F is required for effective down-regulation of TOR during hypoxia and that the failure to down-regulate TOR is sufficient to explain the effect of Ptp61F RNAi on [35S]- methionine incorporation and gene expression under hypoxia.
Ptp61F Governs Persistence of Akt-dependent TOR Activity during Hypoxia
Our results show that Ptp61F negatively regulates TOR activity and that it has a nonredundant role in suppressing TOR function in hypoxia. Work in flies and mammals suggests possible upstream roles of Ptp61F in down-regulating the TOR pathway. PTP-1B, a mammalian homolog of Ptp61F, down-regulates growth factor receptor tyrosine kinase signaling and JAK/STAT signaling (Seely et al., 1996
; Goldstein et al., 1998
; Zabolotny et al., 2002
; Haj et al., 2003
). Although JAK/STAT signaling pathway has not been reported to affect the TOR pathway, we tested its possible contribution by knockdown of JAK or STAT. These depletions did not modify the influence of Ptp61F knockdown on persistence of TOR activity during hypoxia (B). Receptor tyrosine kinases, such as the insulin receptor, activate Akt-mediated phosphorylation of Tsc2, which alleviates TSC inhibition of TOR (Dan et al., 2002
; Inoki et al., 2002
). We hypothesized that Ptp61F may inhibit TOR by interfering with growth factor signaling.
Figure 5. Ptp61F down-regulates phosphorylation of a TOR substrate in hypoxia by suppressing an Akt-dependent signal. (A) Ptp61F is required for efficient down-regulation of insulin signaling under hypoxia. Cells treated with dsRNA against Ptp61F were stimulated (more ...)
To examine this, S2 cells were stimulated with insulin, and insulin receptor phosphorylation, a marker for its activity, was monitored by Western blotting with a phospho-tyrosine specific antibody as well as by its reduced mobility in SDS-PAGE. We assessed the influence of hypoxia and Ptp61F knockdown on receptor activation (A, top two panels). In normoxia, insulin stimulation caused tyrosine phosphorylation and a mobility shift of insulin receptor within 1 h (A, compare lanes 1–3 or 9, Supplemental Figure S2B), and this modification persisted with slight reduction for 16 h (A, lane 7). When cells were stimulated with insulin for 1 h in normoxia and then exposed to hypoxia (<0.1% O2) for 15 h, the tyrosine-phosphorylated band and the mobility shift of insulin receptor disappeared (A, compare lanes 13 and 7), suggesting gradual inactivation of the insulin receptor in hypoxia. Similarly, phosphorylation of Akt, a downstream step in insulin signaling, was almost undetectable after 15 h of hypoxia (A, third panel, lane 7 vs. lane 13). These findings suggest that insulin-dependent signaling is gradually down-regulated during hypoxia.
RNAi knockdown of Ptp61F slightly increased total cellular tyrosine phosphorylation in normoxic cells whether or not they were stimulated with insulin (A, top, lanes 1–8). After 15 h of hypoxia, Ptp61F-deficient cells sustained a high level of tyrosine phosphorylation and substantially retained the slower migrating form of insulin receptor (A, top two panels, lane 13 vs. lane 14). Cells treated with Ptp61F RNAi also displayed sustained Akt phosphorylation (A, middle, lane 13 and lane 14) compared with control cells. These results show that Ptp61F contributes importantly to down-regulation of insulin signaling under hypoxia.
Although Ptp61F operates to down-regulate persistence signaling by added insulin after hypoxia, the above-mentioned experiments did not test whether down-regulation of insulin signaling is the relevant factor in the response examined under normal culture conditions. Indeed, under our normal experimental conditions without added insulin, RNAi against insulin receptor did not abolish the effect of Ptp61F RNAi on TOR activity during hypoxia (B). Because insulin signaling is not required, Ptp61F must operate on alternative or redundant pathways. The insulin receptor is one many receptor tyrosine kinases (rtk). We considered the possibility that various rtk's might provide redundant TOR activation signal and that Ptp61F is a more general inhibitor of rtk signaling. Supporting this possibility, PTP-1B is known to down-regulate other receptor tyrosine kinases in mammalian cells in addition to insulin receptor.
To test the possible role of generic rtk signaling, we knocked down Akt, a common downstream effector of growth factor signaling pathways. To examine the consequence on TOR activity, we assessed p70S6K phosphorylation (C). RNAi of Akt only slightly reduced p70S6K phosphorylation (C, lane 1 vs. lane 3), suggesting that both Akt-dependent and independent activities phosphorylate p70S6K. These results suggest that Akt is a significant, but not the sole, activator of p70S6K phosphorylation in normoxic S2 cells.
The knockdown of Akt did not seem to reduce the already extremely low level of p70S6K phosphorylation in hypoxia (C, lane 5 vs. lane 7). Thus, Akt dependent TOR activity is somehow eliminated during hypoxia. In contrast, knockdown of Akt reduced the phosphorylation of p70S6K that persists in cells under hypoxia when Ptp61F was knocked down (C, lane 6 vs. lane 8), suggesting that a major consequence of reducing Ptp61F is to promote/allow a persistence of Akt-dependent signaling, which activates TOR. These results suggest that Ptp61F acts as a negative regulator of Akt-dependent pathway of TOR regulation whose activity is particularly significant in preventing persistent signaling during hypoxia.
Ptp61F-depleted Cells Are Resistant to Cell Death under Hypoxia
A priori, it is not clear whether inhibition of protein translation during hypoxia will be beneficial or harmful to cell survival. In mammalian cells, lack of Tsc2 prevents efficient down-regulation of translation during hypoxia and allows cells (Tsc2−/−
mouse embryonic fibroblasts) to grow and proliferate under hypoxia (Brugarolas et al., 2004
; Wouters et al., 2005
; Kaper et al., 2006
). We examined this phenomenon in Drosophila
S2 cells and asked whether depletion of Ptp61F has a similar effect on S2 cells under hypoxia.
Cells were treated with control RNAi or Ptp61F RNAi for 4 d, exposed to hypoxia. Cell viability was assessed by FACS after staining with the vital dye Sytox Green, which stains only dead cells. When control RNAi-treated cells were exposed to hypoxia (<0.1% O2), cell viability declined by 3–4 d (A). However, Tsc2 or Ptp61F RNAi-treated cells survived better than control RNAi-treated cells under hypoxia.
Because depletion of Tsc2 or Ptp61F impaired the inhibition of TOR activity under hypoxia, we examined whether TOR activity is responsible for survival of these cells. TOR forms two complexes, mTORC1 and mTORC2 (Sarbassov et al., 2004
; Martin and Hall, 2005
). mTORC1, which includes TOR and Raptor, phosphorylates p70S6K
and 4E-BP1 to promote protein translation (Loewith et al., 2002
). mTORC1 is under the control of the TSC complex, requires Rheb for its activation, and it is inhibited by rapamycin. We confirmed that RNAi against Rheb or Raptor abolished p70S6K
phosphorylation in Drosophila
S2 cells in normoxia (Supplemental Figure S4) (Yang et al., 2006
). Rheb or Raptor RNAi treatment abolished the effect of Ptp61F on cell survival under hypoxia (B), suggesting that TORC1 activity is important in cell survival under prolonged hypoxia treatment in cells lacking Ptp61F. Similarly, the ability of Tsc2 RNAi to improve viability in hypoxia was also eliminated by Raptor or Rheb RNAi (C). These data show that TORC1 activity contributes to the increased cell survival under hypoxia that occurs upon Ptp61F or Tsc2 depletion.