Hepcidin as a Molecular Hub of Iron Homeostasis: From BMP–SMAD Signaling to Therapeutic Modulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an extensive review of the topics, and is potentially interesting to investigators in the field.

Minor points:

Line 105: "with very limited contributions". This might be incorrect, as a 2023 paper by Bigorra Mir confirms high hepcidin expression in the right atrium. Reference should probably be added.

Line 111: The manuscript tends to cite reviews rather than original work. The macroglobulin interaction, although controversial, was first described by Peslova G in 2009. Reference should be added.

Line 115: "and the kidney" seems strange, as previous examples mentions particular cells, not organs. Ferroportin is probably expressed on every cell, including (paradoxically) erythrocytes.

Line 123: Again, original work should be cited (Aschemeyer et al. 2018).

Line 154: Is the current official designation SMAD8  or SMAD9?

Line 166: Please add correct reference to the all-important HFE-TFR2-HJV complex (33?).

Line 178: Reference 33 is a mistake, probably refers to line 166, correct is 34.

Line 185: ERFE binds also BMP5, which might be important for hepcidin regulation (Xiao et al, 2023, PMC10613724.

Line 199: It has NEVER been demonstrated that TMPRSS6 cleaves HJV in vivo. On the contrary, HJV protein is actually decreased in Tmprss6-deficient mice. Please add that the Silvestri reference refers to in vitro results only.

Line 237: Again, the reference includes in vitro work only. Effect of hypoxia on TMPRSS6 protein in vivo has not been demostrated. 

Line 266: The statement "clinical penetrance is incomplete" might be misleading: Actually, the clinical penetrance seems to be almost non-existent, otherwise hospitals would be full with hemochromatosis patients (?). Please elaborate more on this crucial aspect of hemochromatosis pathophysiology.

Line 279: Hepcidin replacement can NOT function as rationale therapy in hemochromatosis, as the patients are typically iron overloaded at the time of diagnosis!  It might function as addition to phlebotomy. Again, please elaborate more on this aspect.

Line 299: As correctly mentioned in Discussion (line 611), the pathophysiology of anemia of inflammation is multifactorial. It is not solely related to hepcidin.

Line 450: Experiments in mice demonstrated that GDF11 is probably not the target of luspatercept (Guerra et al. 2019).

Line 592: Has luspatercept indeed "redefined" thalassemia treatment? Is it a first-choice drug in Italy? If so, it should be mentioned.

 

 

 

 

 

  

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This review article discusses many important aspects of hepcidin, its action, regulation of expression and pathophysiological aspects. After a brief introduction the review focuses mainly on the regulatory pathways of hepcidin expression. There is clear and well-organized description of the different cell signaling pathways involved in iron metabolism regulation. Also, the diseases caused by malfunctioning iron metabolism are presented in a clear, understandable way. Special strength of the paper is the detailed discussion of myeloproliferative diseases. Potential therapeutic considerations are also mentioned with the newest clinical results. Measuring hepcidin level in patients is a not solved question, which is also discussed properly in the paper.

References are up-to-date, figures are very useful in following the text. The organization and the language of the paper are of high quality.

Minor remarks: In 2.1. it should be mentioned that the CNS is special in iron regulation. There is local hepcidin synthesis, which function should be briefly discussed in the paper. In chapter 2. a figure would be nice. In ln 52 humans should be mentioned.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a comprehensive review on hepcidin, iron-metabolism, iron-related disorders and hepcidin-targeting therapies. The information provided is generally up to date; however, the manuscript lacks a specific focus. While the title and early sections suggest a broad review of hepcidin and iron biology, a substantial portion is devoted to myelofibrosis, polycythemia vera, and agents developed in those settings. Consequently, several major areas of hepcidin biology and iron metabolism receive comparatively limited coverage, while peripheral areas are overrepresented. For instance, there is relatively little discussion on hemochromatosis, the prototypic hepcidin-deficiency disorder, both from the mechanistic and the translational point of view. The authors should either revise the title and scope to emphasize MPNs and therapeutic modulation, or expand the discussion of other major hepcidin-related diseases to achieve better balance. Specific issues are outlined below.

• The authors ignore the new classification of hemochromatosis that has been proposed by the BioIron Society (see PMID 34601591).
• The review discusses BMP-SMAD signaling but largely bypasses one of the most important unresolved questions in iron biology: how iron is sensed upstream of hepcidin. The roles of HFE, TFR1 and TFR2 on hepcidin regulation are only superficially covered, and relevant literature is ignored.
• The future perspective section is largely descriptive and lacks a critical discussion on challenges and limitations.
• The authors repeatedly frame the pathway as BMP6-centric. Current understanding favors a broader BMP-SMAD network, with BMP2, BMP6 and BMP2/6 heterodimers contributing in different contexts. In this sense, I would suggest changing “BMP6/ACVR1/SMAD” to “BMP/ACVR1/SMAD”.
• Line 48: “Iron is a paradoxical micronutrient”; this is an awkward statement.
• Lines 62-64: The binding of hepcidin to ferroportin not only triggers ferroportin degradation but also inhibits iron efflux.
• Lines 72-75: Please, note that nutritional immunity is a physiologic response, it does not represent an example of dysregulation of the hepcidin/ferroportin axis.
• Lines 107-108: The systemic contribution of extrahepatic hepcidin appears negligible based on studies in liver-specific Hamp-deficient mice, although local autocrine and paracrine functions remain under investigation.
• Lines 117-121: Hepcidin-mediated degradation of ferroportin is not exclusively executed in lysosomes. There is evidence for proteasomal co-involvement; see PMID 33895792.
• Lines 131-132: The statement “hepcidin insufficiency drives parenchymal iron loading even in the absence of overt exogenous iron loading” is inaccurate. Hepcidin insufficiency does not intrinsically cause systemic iron overload in the absence of iron acquisition. Rather, it promotes enhanced absorption and altered iron distribution, which under normal dietary conditions eventually result in iron overload.
• Lines 145-147: It should be indicated that BMP2 and BMP6 also act as heterodimers.
• Lines 183 and 196: Reference [35] is irrelevant to the statements.
• Lines 198-201: The model that matriptase-2 mitigates hepcidin expression by cleaving Hjv and other components has been challenged by data from Caroline Enns’ lab showing that catalytically inactive matriptase-2 mutants retain their hepcidin-suppressor function.
• Line 226: Anemia of inflammation is often termed anemia of chronic disease. However, the term functional anemia is uncommon.
• Line 267: Reference [33] is irrelevant to the statement.
• Paragraph 4.2: The first sentence describes anemia of inflammation as “mechanistically hepcidin-driven anemia”, while the last sentence highlights the “complex” multifactorial nature” of inflammatory anemias. In fact, inflammatory induction of hepcidin is one of many other contributors to anemia.
• Lines 401-403: A reference to support the statement is missing.
• Lines 420-422: Anemia of inflammation is not microcytic (at least if not combined with true iron deficiency).
• Lines 476-479: A reference to support the statement is missing.
• Lines 128 and 257: Please, correct typos.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript is improved, and the authors have appropriately responded to most queries. However, there are remaining issues that need to be addressed.

• Lines 227-238. The authors describe a “current” model with a reference from 1999. The interactions between TFR1, HFE, TFR2 and HJV are presented in an oversimplified way. The current state of knowledge can be deducted from more recent publications (such as PMID 36322932, 41662592, 40913261) and editorials (such as PMID 36701171, 42096253).
• Lines 238-241. There is significant progress on understanding how iron induces BMP6 expression in liver sinusoidal endothelial cells, which is not captured in this description. Relevant publications are PMID 31276102, 36351237, 39437541, 40561335; see also accompanying editorials (PMID 32694854, 36656611, 39913338, 41037298).
• Line 254. Ref. 44 is irrelevant here; it should be replaced by PMID 30097509 and 31800957.
• Line 272. Only ref. 50 is supportive to the statement.
• Line 275. Ref. 50 is wrong here; the correct reference is PMID 21612955.
• Lines 361-371. While the content of the paragraph is correct, there is no information on recent GWAS studies demonstrating that individuals carrying the C282Y variant are at increased risk of several disorders including liver cancer, other liver complications, musculoskeletal morbidity, general morbidity and infectious diseases (PMID 33231665, 39178373, 37808392, 38479735, 38728387).
• Line 371. Ref. 40 is irrelevant here.
• Lines 28 and 84. Please, replace BMP6 with BMP.
• Line 76. Please, move “and” before “the iron-restrictive anemia of myelofibrosis”.

Author Response

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Author Response File: Author Response.pdf