Sabina Alam, PhD
Editor
BMC Medicine
Nicolas C. Nicolaides, MD, PhD
Division of Endocrinology and Metabolism
Clinical Research Center
Biomedical Research Foundation of the Academy of Athens
4 Soranou tou Efessiou Street
Athens, 11527, Greece
Tel: +30 – 210 – 6597041
Fax: +30 – 210 – 6597545
E-mail:
nnicolaides@bioacademy.gr
December 19, 2013
Dear Professor Alam,
Re: MS: 7190576501053833-Recent Advances in the Molecular Mechanisms
Determining Tissue Sensitivity to Glucocorticoids: Clinical Implications
Evangelia Charmandari, Nicolas C. Nicolaides, George P. Chrousos and Tomoshige
Kino.
BMC Medicine
Thank you very much for your letter dated October 17, 2013 and the comments of the
Reviewer. We have responded to these comments fully and have revised our manuscript
according to the Reviewer’s suggestions. We hope that it is now suitable for publication
in BMC Endocrine Disorders.
The responses to the comments of the Editor-in-Chief and the Reviewers, and the
revisions made are as follows: (Comments are in bold font style and our Responses in
regular font style):
Editor’s-in-Chief Comments:
1. The wider literature has not be fully discussed which will be important to do in
order to provide a balanced review. Therefore, you should ensure you cite other
groups that have researched GC sensitivity. This should particularly in the
introduction.
In response to your comment, we have enriched the part of background by citing
important findings from other groups, since this was also suggested by the Reviewer
(Please see our responses for the Reviewer’s comments 1-4). For this purpose, we have
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added 19 new references and have expanded the length of our manuscript from 2323 to
2886 words.
2. State explicitly that you have focused on three aspects of GR signalling: circadian
timing and GR mutations, and homologous downregulation, (rather than a general
overview of recent advances in GR sensitivity).
We have stated clearly that our review has focused particularly on three aspects of GR
signaling: novel mutations, circadian rhythmicity and ligand-induced repression of hGR.
This is explicitly stated in three parts of the manuscript: 1. Title (Page 1, lines 8-10), 2.
Abstract (Page 3, lines 14-15) and 3. Background (Page 7, lines 11-13).
3. Remove any overlapping text with current literature.
We have removed any overlapping parts of text with previously published reviews.
Reviewer’s Comments:
In this review, the authors discuss the most recent findings regarding the
mechanisms which regulate the sensitivity of different tissues to glucocorticoids.
They particularly focus on mutations of the hGR gene that can cause Primary
Generalized Glucocorticoid Resistance, the effects of circadian rhythmicity on
glucocorticoid sensitivity and ligand-induced repression of hGR expression.
This review is well-written, informative and considerably improved compared with
the previous version, limiting overlaps with previously published reviews. However
there are some issues that must be taken care of in order to improve the quality of
the review.
We thank the Reviewer for his/her comments and we have revised our review according
to his/her suggestions.
Major compulsory revisions
1. Although the authors promise an account of the recent advances in the molecular
mechanisms determining tissue sensitivity to glucocorticoids, they overlooked some
important recent findings regarding components of GR-signaling such as the role of
SGK-1 (Anacker et al., PNAS, 2013; Schwab et al., Cellular Physiology and
Biochemistry, 2008), FKBP5 (Menke et al., Genes, Brain and Behavior, 2013;
Hartmann et al., Neuropharmacology, 2012) or coregulator expression and
recruitment (Bender et al., Molecular Endocrinology 2013; Lachize et al., PNAS,
2009). These findings indicate that these components may be equally important to
GR-signaling as the mutations of GR and may have clinical implications as well.
Focusing on certain factors dictating glucocorticoid sensitivity is not negative or
undesirable per se, however, one would expect, at least, brief references to recent
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findings regarding other players involved in GR-signaling. Perhaps something like
this can be added in the Background section of the review.
We agree with the Reviewer and we have added brief references to important findings for
molecules involved, directly or indirectly, in GR signal transduction and associated
pathologic conditions. Regarding SGK1 and FKBP5, please see page 6, line 24, and page
7, lines 1-3. As for coregulator expression and recruitment, please see page 5, lines 12-14
and 22-23. All these parts of the text have been added in the Background section, as
suggested by the Reviewer.
2. Another component of GR-dependent signaling pathways is the membrane bound
GR which modulates the non-genomic effects of glucocorticoids (Groeneweg et al.,
Journal of Endocrinology, 2011).
We thank the Reviewer for his/her comment. We have added a brief part of text
discussing the membrane GR-mediated non-genomic glucocorticoid actions (Page 6,
lines 13-15). These findings are cited in Groeneweg et al., Journal of Endocrinology,
2011, as suggested by the Reviewer.
3. Apart from the mentioned mutations that decrease the sensitivity of GR, other
splice variants exist which may lead to enhanced responses depending on the
glucocorticoid used (e.g.; Baker et al, Shock, 2012; Tung et al., Shock, 2011) and the
authors include one or two of them in table 3. Discussing this issue may increase the
impact of this review, as no rationale is provided for choosing to discuss only
mutations that decrease sensitivity to glucocorticoids. Where is the work on the GR
polymorphisms??
The recently identified splice isoforms of GR are briefly reported in page 6, lines 21-22.
Regarding GR polymorphisms (page 7, lines 3-6), we have added the references 20-24.
4. Glucocorticoid signaling may also be mediated by the other receptor of
glucocorticoids, the mineralocorticoid receptor. The existence of two receptor
system for glucocorticoid signaling must be at least acknowledged, as
polymorphisms of MR may also play a role in glucocorticoid sensitivity (van
Leeuwen et al., Psychoneuroendocrinology, 2011,) especially since GR and MR show
overlapping expression patterns in many tissues and may also share common target
genes.
The involvement of MR in glucocorticoid signaling is reported in page 6, lines 15-18.
We have also discussed briefly the important role of MR polymorphisms by citing van
Leeuwen et al., Psychoneuroendocrinology, 2011 in page 7, lines 6-7, as suggested by the
Reviewer.
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5. The previous four points combined may point out that the title of the review may
not be an accurate reflection of the content of the review, since a few of the
mechanisms that determine tissue sensitivity to glucocorticoids have been
completely ignored by the authors.
We thank the Reviewer for his/her comment. We have revised the title of our review as:
“Recent Advances in the Molecular Mechanisms Determining Tissue Sensitivity to
Glucocorticoids: Novel Mutations, Circadian Rhythm and Ligand-induced Repression of
the Human Glucocorticoid Receptor”.
6. The authors describe in detail the interactions between CLOCK and hGR. This
begs the question whether anything is known about the interactions between
CLOCK and mutated forms of GR. Also, regarding the interplay between tissue
glucocorticoid sensitivity and circadian rhythmicity, it might be valid to briefly
discuss the effects of pulsatility of glucocorticoid secretion on tissue sensitivity to
glucocorticoids.
To the best of our knowledge, any interactions between CLOCK and mutant forms of GR
have not been investigated.
Regarding the interplay between tissue glucocorticoid sensitivity and circadian
rhythmicity, we have briefly discussed ultradian cortisol pulsatility in page 9 (lines 5-10).
7. The figures are of excellent quality, but it should be explicitly mentioned that they
have been used in previous publications.
We have prepared new figures (figures 1-3), which have not been published.
Minor issues:
1. Pathologic natural hGR mutations causing Primary Generalized Glucocorticoid
resistance or Chrousos syndrome, third paragraph: It may be beneficial to add
references ?of the previously described differential effects of hGR mutations?? in
the end of the paragraph.
Amended as suggested by the Reviewer (Page 8, lines 22-23).
2. Ligand-induced down-regulation (repression) of hGR gene expression: Is it
known how the histones and/or the DNA are modified at the promoter region of the
hGR after treatment with glucocorticoids?
To the best of our knowledge, it is not known how the histones and/or the DNA are
modified at the promoter region of the hGR after treatment with glucocorticoids.
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Please, find attached:

The file containing the text, references and figure legends of the manuscript.

The file containing the Figures.
Please, do not hesitate to contact me if you require any further information.
I look forward to hearing from you.
With best wishes,
Yours sincerely,
Nicolas C. Nicolaides, MD, PhD
Postdoctoral Fellow
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