Dear editors:
Manuscript ID JCB-10-0254 entitled "VEGF stimulation enhances Livin protein
synthesis through mTOR signaling" has been revised according to the reviewers’
suggestions. A rebuttal letter is also provided in which we respond to each point raised by
each reviewer. Moreover, we add the research content about Livin expression regulation
in melanoma model as recommended by Reviewer 3. Additionally, we ask a native
speaker to revise our English expression to make the results clear and well presented.
Response to reviewer’s suggestion
Reviewer: 1
1. Figure 1B shows no increase in expression of Livin-protein after treatment with
VEGF. It is certainly a mislabelling, but both in text and figure legend authors are
suggesting that on this figure the effects of three growth factors are shown. However,
they present only two blots. The not shown data should be clearly declared.
Thanks for your remind. There are two mislabeling in Figure 1B. We have conducted
corresponding revision. We feel very sorry for our mistake.
2. Does VEGF induce livin expression in a concentration-dependent manner? This
question should be addressed by the authors.
Yes, VEGF can induce livin expression in a concentration-dependent manner.
To answer this, we treated HeLa cells with various concentrations of VEGF for 15 h. The
expression of livin was determined by Western blots. As shown in the below figure, Livin
protein levels were significantly induced in a dose-dependent manner, with maximum
induction at 50ng/ml. Quantitative analysis reveals that there is no significant difference
between 10 ng/ml and 50ng/ml. This content has been added in the manuscript.
VEGF (ng/ml)
0
0.1
1
10
50
Livin
β-actin
3. The Figures 1a and 3c show experiments done under similar conditions. In Figure
3c is shown that (after 15h of treatment), the livin expression is induced more
strongly by FBS than VEGF. Figure 1A exhibits the opposite. Which experiment is
representative? The authors should explain this discrepancy.
Thanks for your question. We performed the experiment again, and the result showed that
livin expression is induced more strongly by VEGF than FBS treatment. Thus figure 1A
is the representative figure. I think the discrepancy is caused by different exposure times
in Western blots. Initially, the two experiments were conducted by two different people.
Now, the repeated experiment was performed by one guy. The exposure time in Western
blots was tightly controlled. Figure 3a (15h treatment) has been revised. Finally, we
apologize for this error, and appreciate for your careful review.
4. Figure 4B should include descriptions on x-axis. Namely, the order of bars is not
clear: is it as on panel A or as in figure legend. This lack of clarity should be
eliminated.
We have added descriptions on x-axis in figure 4B. We hope it can eliminate the clarity
in the figure.
5. Figure 5: The title is misleading.
Yes, the original title is misleading. We have changed the title to “4E-BP1 is involved in
regulation of Livin expression”.
6. Figure 5B: The comparison of Livin expression after VEGF treatment and 4EBP1-silencing (in otherwise untreated cells) should be performed. This experiment
could specify if 4E-BP-1 is the only or crucial effector in regulation of livin
expression by VEGF/mTor; or other potential factors play a role.
We have compared livin expression in VEGF-treated cells and 4E-BP1-silencing cells
without VEGF-treated. The result showed that 4E-BP1-silencing can substitute VEGF
stimulation in inducing livin expression in serum-deprived HeLa cells, suggesting 4EBP1 is the only effector in regulation of livin expression by VEGF/mTOR. The section
has been added in the manuscript.
7. Discussion: some interesting results are not sufficiently discussed (e.g. selection of
the VEGF phosphorylation target).
We have extended our discussion about some interesting results.
Reviewer: 2
Major points:
- Do the authors have indications that regulation of the Livin-protein level is by
VEGF is not HeLa cell specific?
VEGF-mediated livin induction is not HeLa cell specific, but the effect is involved in
other cells with livin expression. The research about livin regulation has also performed
in melanoma model, and the related results have been added in manuscripts.
- the authors demonstrate reduced cell viability on combined treatment with
doxorubicin and inhibitors of the mTOR-pathway? At the same time they
demonstrate that inhibitors of the mTOR-pathway result in reduced Livin protein
levels. Even though these data are highly suggestive that deregulation of Livin
contributes to the enhanced cytotoxic effect of doxorubicin, the authors should
directly demonstrate that Livin is the responsible factor (e.g. using siRNA against
Livin) and not other factors deregulated by VEGF/rapamycin.
To answer this question, we used siRNA knockdown to inhibit Livin expression. The
result showed that Livin silencing cells have higher caspase3/7 activity and lower cell
viability than untreated cells in response to doxorubicin addition, but they do not show
significant difference from the cells whose mTOR signaling pathway is inhibited. The
result has been added in the manuscripts.
The authors use the MTT-assay (proliferative status) as a functional endpoint. Do
the authors have any indications for enhanced amounts of apoptosis (or other modes
of cell killing) in response to the different treatment regimens?
Thanks for your question. To determine apoptosis degree is correlated with Livin levels,
we also assay another apoptosis-related indictor, caspase3/7 activity, using Promega kit.
The result suggested that exposure of serum-deprived HeLa cells to doxorubicin resulted
in significant increase in caspase3/7 activity. But the increase is abolished by VEGF
stimulation but not by rapamycin plus VEGF treatment. Further, Livin silencing cells
have higher caspase3/7 activity than untreated cells in response to doxorubicin addition.
Minor points:
- Fig 1 B: wrong labeling?
The wrong labeling has been revised.
- Fig 2B: could be omitted
As the suggestion, we have transferred figure 2B to supplementary materials in the
revised manuscript.
- The authors claim that 4E-BPI is a main regulator of Livin expression: however
other elements of this pathway maybe as important - please clarify
Regulation of protein synthesis involves a number of different possible mechanisms
acting on initiation and/or elongation steps of mRNA translation. Recruitment of mRNA
to ribosomes in mammalian cells involves interaction of the 5’m7GpppN (cap) structure
on the mRNA with initiation factor eIF4E, also known as cap-binding protein. eIF4E is a
component of the cap-initiation complex, a group of interacting proteins that bridge
mRNA, the ribosome, and the initiation machinery to initiate protein synthesis. The capinitiation complex contains the scaffold protein eIF4G, which binds eIF4E; the ATPdependent RNA helicase known as eIF4A; a multi-subunit factor known as eIF3 that
binds the 40S ribosomal subunit; and the poly(A)-binding protein (PABP), which
stimulates translation (Gingras et al,1999).
4E-BP1, the major member of eIF4E-binding protein family, binds eIF4E and
competitively inhibits its association with eIF4G, preventing formation of the capinitiation complex and reducing cap-dependent mRNA translation (Gingras et al, 1999).
Hyperphosphorylation of 4E-BP1 carried out by mTOR inhibits 4E-BP1 binding to
eIF4E,
thereby
promoting
protein
synthesis
(Hay
&
Sonenberg,
Hyperphosphorylation of 4E-BP1 carried out by mTOR inhibits 4E-BP1 binding to
2004).
eIF4E, thereby promoting protein synthesis (Hay & Sonenberg, 2004). Through Western
blots analysis, we found that VEGF treatment could enhance 4E-BP1 phosphorylation at
all sites tested. Hyperphosphorylation of 4E-BP1 will in turn promote protein synthesis.
The elongation step of protein synthesis is also subject to inhibition by
phosphorylation of eukaryotic elongation factor 2 (eEF2) at position Thr-56. eEF2
mediates
ribosomal
translocation
during
peptide
chain
elongation.
eEF2
is
phosphorylated by eEF2 kinase (eEF2K), which is both inhibited and activated by
phosphorylation at a number of sites (Browne & Proud,2002). In the study, we did not
detect significant changes in the phosphorylation state of eIF2-α upon VEGF stimulation,
thus we ruled out its effect on Livin protein synthesis.
In mammalian cells, mTOR positively controls protein synthesis, which is required for
cell growth, through various downstream effectors. mTOR promotes protein synthesis by
phosphorylating the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1)
and the p70 ribosomal S6 kinase 1 (S6K1).
However, in the study VEGF treatment did
not change the phosphorylation state of p70 S6K1. Thus, we also did not consider
p70S6K effect on Livin protein expression although the stimulation of S6K1 activity by
mTOR leads to increases in mRNA biogenesis, cap-dependent translation and elongation,
and the translation of ribosomal proteins through regulation of the activity of many
proteins, such as S6K1 aly/REF-like target (SKAR), programmed cell death 4 (PDCD4),
eukaryotic elongation factor 2 kinase (eEF2K) and ribosomal protein S6 (Ma and Blenis,
2009).
- the manuscript should be carefully checked for language aspects
The manuscript has been checked by a native speaker. I hope it can meet the requirement.
Reviewer:
3
Specific comments:
i) The authors suggest that VEGF-induced upregulation of Livin is specific and
indeed VEGF does not induce XIAP. It is of importance to examine the effect of
VEGF stimulation on the protein level of Survivn that has been shown by Vaira et al
2006 (also cited in this manuscript) to be induced by IGF, also through the mTOR
pathway, however by p70S6K. The biological relevance of VEGF-induced
upregulation of Livin is not clear because of the cell culture model used, serumdeprived HeLa cells. It is important to evaluate the effect of VEGF on Livin in a
more relevant model, e.g. melanoma cells. Furthermore, it should be examined, in
melanoma (or other) cells that express Livin in the absence of VEGF, whether
inhibition of the mTOR pathway will inhibit Livin protein expression. This is of
great importance as it can indicate that such inhibition will result in better response
to chemotherapy of Livin positive tumors.
Thanks for your question. As the suggestion, we conducted relative experiment in SKMEL-28 cells, a melanoma cell lines. Western blots analysis showed that Livin protein is
constitutively expressed in SK-MEL-28 cells. Prolonged exposure of SK-MEL-28 cells
to the mTOR inhibitor rapamycin caused a marked decrease in Livin expression.In
addition, we also found that VEGF treatment could lead to increased expression of Livin
protein, but the induction was abrogated by mTOR inhibition.
ii) The results describing the "regulation of cell viability by mTOR / Livin
signaling", presented in figure 6. The results in Figure 6C are interesting and,
importantly, as the authors suggest "modulation of VEGF/mTOR signaling
combined with cytotoxic treatment provides a novel strategy for cancer treatment".
However, regarding Livin, the expression of Livin in these experiments is consistent
with the results shown in Figures 1 and 3 and the sensitivity of cells to apoptosis
induced by cytotoxic agents such as doxorubicin is known to correlate with Livin
protein level. Perhaps this section should be edited to better explain the findings
that once again correlate Livin protein level, regulated by VEGF and cells death
induced by cytotoxic agents.
We have re-edited this section in the revised manuscript. To further verify the finding, we
directly demonstrate that deregulation of Livin contributes to the enhanced cytotoxic
effect of doxorubicin using siRNA against Livin. Furthermore, we also select another
apoptosis-related indicator to confirm that livin expression level is associated with the
apoptosis degree in response to cytotoxic agents.
iii) How reproducible is the induction of Livin by VEGF as demonstrated in Figure
1A (in Figure 3A the induction is much lower)?
To answer this question, we performed the experiment again, and the result showed that
livin expression is induced more strongly by VEGF than FBS treatment. Thus figure 1A
is the representative figure. I think the discrepancy is caused by different exposure times
in Western blots. Initially, the two experiments were conducted by different people. The
repeated experiment was performed by one guy. The exposure time in Western blots was
tightly controlled. Figure 3a (15h treatment) has been revised. Finally, we apologize for
this error, and appreciate for your careful review.
iv) Figure 1B, top stimulation is probably not VEGF (EGF?) – should be corrected.
The wrong labeling has been revised.
v) Figure 4B – need to label X-axis (also in D and F though less important).
We have added descriptions on x-axis in figure 4B, D and F. We hope it can eliminate the
clarity in the figure.
vi) Figure 6 - correct legend of fig 6 to explain that 6A=15hr and 6 C=30hr.
Thanks for your suggestion. The figure legend has been revised.
vii) The model in Figure 7 should be edited – in the current version, mTOR seems to
inhibit phosphorylated 4E-BP1. It should better present that mTOR phophorylates
4E-BP1 and thereby inhibits 4E-BP1 activity
The model has been edited. Current model shows that VEGF stimulation activate mTOR
signaling, enhance the phosphorylation levels of TOR (Ser-2448), and then 4E-BP1 is
hyperphosphorylated thereby inhibits the activity of 4E-BP1. Dissociation of 4E-BP1
promotes to eIF4E release, and in turn promotes livin protein synthesis.
viii) The article should be revised by a native English speaker scientist.
The manuscript has been checked by a native speaker. I hope it can meet the requirement.