Development of a Novel VDR-Activating Peptide as a Functional Cosmetic Ingredient for Skin Barrier Health and Photoprotection

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

 Point-by-point response to Comments and Suggestions for Authors

Comments 1:

The manuscript frequently refers to VDR‑Pep as a “novel” VDR agonist. While the approach is interesting, the novelty is not sufficiently contextualised. The Introduction would benefit from a clearer comparison with existing vitamin D analogues and previously reported strategies aimed at modulating VDR activity for cosmetic applications. Explicitly stating how this peptide differs, either structurally or functionally, from existing approaches would strengthen the scientific positioning of the work.

Response 1:

Thank you for pointing this out. We agree with this comment. Therefore, we have revised the Introduction to better contextualize the novelty of VDR-Pep by explicitly comparing it with existing vitamin D analogues and previously reported strategies for modulating VDR signaling. Specifically, we have clarified how the peptide-based approach differs from conventional steroidal vitamin D derivatives in terms of structure and mechanism of action, and highlighted its potential advantages for cosmetic applications, including improved photostability and reduced systemic activity.

This revision has been incorporated in the Introduction section (Page 02, Lines 60-64, 65-68).

“In response to these limitations, various strategies have been explored to modulate VDR signaling, including the development of synthetic vitamin D analogues and non-calcemic derivatives. However, these approaches still largely rely on steroidal structures and may retain concerns related to biological activity and formulation stability”

“approach: designed to engage the ligand-binding domain of VDR without relying on hormonally active compounds, such peptides offer improved photostability, formulation compatibility, and a more favorable regulatory profile”

Comments 2:

The central claim of the manuscript is that VDR‑Pep acts as a VDR agonist. This conclusion is mainly supported by increased VDR/RXR heterodimerization and downstream marker expression. However, receptor dependency is not formally demonstrated. No VDR knockdown, antagonist, or inhibition studies are included to confirm that the observed effects are strictly VDR‑mediated. As a result, the mechanistic interpretation should be handled more cautiously. The authors are encouraged either to moderate their terminology (e.g., “VDR‑associated activation”) or to explicitly acknowledge this limitation in the Discussion.

Response 2:

Thank you for this important comment. We agree that direct receptor dependency was not formally demonstrated in the current study. Therefore, we have revised the manuscript to moderate the interpretation of VDR-Pep as a definitive VDR agonist and to acknowledge this limitation more explicitly.

Specifically, we have adjusted the terminology in relevant sections to reflect a more cautious interpretation :”VDR-activating peptide” and we have added a statement in the Discussion to clarify that receptor dependency has not yet been confirmed through knockdown or inhibition approaches.

These revisions have been incorporated in the Discussion section (Page 13, Lines 506-509).

“Although VDR-Pep enhanced VDR/RXR heterodimerization and downstream signaling markers, direct evidence of receptor dependency was not established in this study. Future studies employing VDR knockdown or pharmacological inhibition will be required to confirm whether the observed effects are strictly mediated through VDR.”

Comments 3

The involvement of NRF2 signaling is inferred from enhanced NRF2–RNA polymerase II interactions detected by PLA. While this is an interesting and potentially relevant observation, it remains an indirect indicator of NRF2 pathway activation. The absence of downstream antioxidant gene expression analysis (such as HO‑1, NQO1, or GCLC) should be discussed more clearly as a limitation. Without this clarification, the conclusions regarding NRF2 activation may appear overstated.

Response 3:

Thank you for this insightful comment. We agree that the current data provide indirect evidence of NRF2 pathway involvement and do not fully establish functional activation of downstream antioxidant responses. Therefore, we have revised the Discussion to more clearly acknowledge this limitation and to moderate the interpretation of NRF2 activation.

This revision has been incorporated in the Discussion section (Page 11, Lines 539-543).

“However, the absence of downstream antioxidant gene expression analysis (e.g., HO-1, NQO1, GCLC) represents a limitation. Therefore, the current results should be interpreted as indicative of NRF2-associated transcriptional engagement rather than definitive activation of the NRF2 pathway”

Comments 4:

VDR‑Pep concentrations are reported in ppm, but their relevance to cosmetic formulations or realistic topical exposure is not discussed. A short justification or contextual comparison with concentrations typically used for cosmetic peptides in vitro would improve the translational value of the study.

Response 4:

Thank you for this valuable suggestion. We agree that the relevance of the peptide concentrations used in this study should be better contextualized in relation to cosmetic applications. Therefore, we have added an explanation to clarify that the selected concentration range is consistent with commonly used in vitro concentrations for cosmetic peptides.

This addition has been incorporated in the Materials and Methods (or Discussion) section (Page 03, Lines 179-182).

The concentrations of VDR-Pep (1–200 ppm) used in this study are within the range commonly employed for cosmetic peptides in in vitro assays. These concentrations were selected to ensure detectable biological responses while remaining within a physiologically relevant range for topical applications.

Comments 5:

The statistical analysis section should specify post hoc tests used following ANOVA and clearly indicate the number of biological replicates.

Response 5:

Thank you for this comment. We agree that the statistical analysis should be described more clearly. Therefore, we have revised the Methods section to explicitly specify the post hoc test used following ANOVA and to indicate the number of biological replicates.

This revision has been incorporated in the Statistical Analysis section (Page 04, Lines 264-271).

“All experiments were performed with at least three independent biological replicates. Data are presented as mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism (version 4.0; GraphPad, San Diego, USA). Differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.”

Comments 6:

Minor typographical and formatting issues are present throughout the manuscript and should be addressed during revision.

Response 6:

Thank you for this suggestion. We have carefully reviewed the entire manuscript and corrected minor typographical, grammatical, and formatting errors to improve clarity and consistency throughout the text.

Comments 7:

The Introduction, while informative, could be slightly condensed to improve readability.

Response 7:

Thank you for this helpful suggestion. We have revised the Introduction to improve readability by condensing repetitive or less essential content while preserving the key background and scientific rationale of the study.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

* Point-by-point response to Comments and Suggestions for Authors

Comments 1

Typographical and Style Issues

Line 79: in vitro should be written in italics.

Line 221: via should be written in italics.

Line 306: in situ should be written in italics.

Line 334: in vitro should be written in italics.

Punctuation and Conventions

Line 247: The period should be placed after the reference → [6]. instead of before.

Line 299: “western blot” should not be capitalized.

Response 1:

Thank you for the careful review. The indicated typographical and style issues have been corrected throughout the manuscript.

Comments 2

Figures (Major Issues)

Figures 1, 2, 3, 4, and 7:

The x-axis is not clearly labeled.

Units should not be written directly in the axis titles; they should appear in parentheses or be described in the figure legends.

Figures 5 and 6:

Error bars are missing, which limits the interpretation of the data.

Please include:

error bars (SD or SEM)

number of replicates (n)

statistical test information (in the figure or legend)

Response 2:

Thank you for these helpful suggestions. We have revised Figures 1, 2, 3, 4, and 7 to improve x-axis labeling clarity and adjusted the unit presentation format according to the reviewer’s recommendation. Units are now presented in parentheses or described in the corresponding figure legends where appropriate.

For Figures 5 and 6, statistical information including the number of independent experiments (n) and the statistical analysis method has been clearly specified in the figure legends. Error bars representing standard deviation (SD) were added where appropriate to improve data interpretation and consistency across figures.

Comments 3

Discussion (Major Issue)

The Discussion section does not contain any references, which is unusual and scientifically inadequate.

It is strongly recommended to:

include relevant and recent references on:

VDR function in skin biology

peptide-based agonists

VDR–NRF2 interactions

compare your findings with existing literature

avoid a purely descriptive discussion (currently too close to the Results section)

Scientific Rigor

Clearly specify:

the number of biological replicates

data normalization methods

Clarify statements where causality is implied without direct evidence (e.g., VDR → NRF2 relationship)

Structure and Clarity

Some sentences are overly long and should be simplified for better readability.

Reduce redundancy between the Results and Discussion sections.

Response 3:

Thank you for this important comment. We agree that the Discussion section required stronger integration with the existing literature and more mechanistic interpretation of the findings. Therefore, we have substantially revised the Discussion by incorporating recent and relevant references related to VDR function in skin biology, peptide-based receptor modulation, and potential interactions between VDR and NRF2-associated signaling pathways. In addition, we have revised several sections to better compare our findings with previously reported studies and to reduce descriptive overlap with the Results section.

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

*Point-by-point response to Comments and Suggestions for Authors

Comments 1:

The abstract should include recommendations for future studies.

Response 1:

Thank you for pointing this out. We agree with this comment.

This revision has been incorporated in the abstract section(Page 1, Lines 30-32).

“Future studies should focus on validating these effects in in vivo human skin models, assessing long-term safety and efficacy, and optimizing formulation stability for practical cosmetic applications.”

Comments 2

The references consulted to determine the cell viability test should be specified. Furthermore, the basis on which the VDR-Pep concentrations were selected should be explained.

Response 2

Thank you for this valuable comment. We have revised the manuscript to clarify the rationale for selecting the VDR-Pep treatment concentrations. Specifically, we added an explanation that the tested concentrations were determined based on preliminary cytotoxicity evaluations, dose-dependent efficacy observations, and their practical applicability in cosmetic formulations.

This revision has been incorporated in the Materials and Methods section (Page 3, Lines 210-212).

“The concentration range used for the cell viability assay was determined based on previously reported peptide treatment conditions in HaCaT keratinocytes and preliminary optimization experiments”

Comments 3

The Western blot analysis has been described in detail. However, the reference according to which this method was performed should be specified.

Response 3

Thank you for the helpful comment. We have revised the manuscript to include appropriate references for the Western blot procedure. The method was performed according to previously established protocols with minor modifications.

This revision has been incorporated in the Materials and Methods section (Page 04, Lines 236).

“Western blotting was performed as described previously [19]”

Comments 4

Detailed information regarding the experimental design of the study should be provided.

Response 4

Thank you for this constructive comment. We have revised the manuscript to provide more detailed information regarding the experimental design, including treatment conditions, concentration ranges, incubation times, number of independent experiments, and statistical analysis procedures. In addition, the methodological descriptions for each assay were clarified to improve reproducibility and overall transparency of the study design.

Comments 5

In the Results section, state whether the treatments affected the characteristics investigated. Also, the phrase (p<0.05) should be added after the phrase “statistical differences.

Response 5

Thank you for the valuable comment. The Results section already describes the effects of the treatments on the investigated parameters, including increases or decreases in marker expression and inflammatory responses. To improve clarity, we carefully reviewed and slightly revised the relevant descriptions throughout the Results section. In addition, “(p < 0.05)” has been added where statistical differences are described in statistical analysis section.

Comments 6

In all figures, superscript lettering should be used to indicate statistical differences. Some tables are missing letter annotations; please complete these accordingly.

Response 6

Thank you for the valuable suggestion. In the present study, statistical comparisons were primarily performed against the untreated control group rather than among all treatment groups. Therefore, significance was indicated using asterisk notation (*p < 0.05 vs. control), which is a commonly accepted method for control-based comparisons and provides clearer visualization for the current experimental design. We have, however, carefully reviewed all figures and ensured that statistical annotations are presented consistently throughout the manuscript.

Comments 7

The resolution of Figure 5b could be improved.

Response 7

Thank you for the helpful comment. Figure 5b has been replaced with a higher-resolution version to improve image clarity and overall visual quality.

Comments 8

In the Discussion section, the findings are well interpreted but not sufficiently discussed in relation to the relevant literature. This section should be revised. “VDR‑associated activation”) or to explicitly acknowledge this limitation in the Discussion.

Response 8

Thank you for this constructive comment. The Discussion section has been substantially revised to strengthen the connection between our findings and previously published literature. Additional discussion was incorporated regarding the established roles of VDR signaling in keratinocyte differentiation, barrier-associated protein expression and NRF2-associated antioxidant pathways. We also expanded the comparative discussion highlighting the potential advantages of peptide-based VDR modulation over conventional vitamin D derivatives for cosmetic applications.

This revision has been incorporated in the Discusstion section (Page 10, Lines 475, 484-487,489-493,521-522,540-543).

Consistent with previous reports demonstrating that VDR activation promotes keratinocyte differentiation,

Reduced expression of filaggrin, involucrin, and loricrin has been closely associated with impaired epidermal barrier function and inflammatory skin disorders such as atopic der-matitis. Therefore, the upregulation of these proteins by VDR-Pep suggests potential rele-vance for barrier-restorative cosmetic applications.

Several studies have suggested functional crosstalk between VDR signaling and NRF2-mediated antioxidant pathways in maintaining cutaneous redox homeostasis.

Unlike conventional vitamin D derivatives, which are often limited by photoinstability and formulation constraints, peptide-based VDR modulators may provide a more stable and cosmetically adaptable approach for regulating epidermal homeostasis.