Surfactin Inhibits Osteoclast Differentiation by Negatively Regulating the Elk1-AP-1-NFATc1 Axis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled "Surfactin inhibits osteoclast differentiation by negatively regulating the Elk1-AP-1-NFATc1 axis" is timely and novel. However, there are several concerns that need to be addressed to strengthen its scientific rigor.

Major Concerns

1. The authors conclude that surfactin inhibits the Elk1–AP-1–NFATc1 pathway, though several mechanistic gaps still exist as follows:

• Elk1 is a MAPK-dependent transcription factor, yet the authors claim MAPK is unchanged. Therefore, how does Elk1 phosphorylation decrease without upstream pathway suppression?
• The study does not investigate CREB, RSK, CaMK, or other signaling nodes upstream of Elk1, even though they are mentioned in the Discussion.

2. The Discussion states that the surfactin used contains multiple analogs with different fatty-acid chain lengths and branching patterns, which may explain discrepancies with prior studies. However, the authors did not show any comparison with single-analog surfactin. As well as no examination of dose–response for individual analog classes.

 

3. The manuscript reports NFκB and MAPK signaling were analyzed at a single time point (15 min). However, RANKL signaling is dynamic, and some regulators show biphasic responses.

 

4. The author should measure the phospho and total NFκB (p65) levels in both cytoplasm and nucleus to determine the activation of NFκB. Additionally, the Western blot images (Figures 4-6) lack quantification.

 

Minor Concerns

1. TRAP and F-Actin Images Need Higher Resolution
2. The manuscript does not specify whether normality tests were performed. The sample size (n) is not stated in the figures. Whether experiments were repeated independently is unclear.
3. Some sentences in the Introduction and Discussion are overly long and should be streamlined.

Author Response

Major Concerns

1. The authors conclude that surfactin inhibits the Elk1–AP-1–NFATc1 pathway, though several mechanistic gaps still exist as follows:

• Elk1 is a MAPK-dependent transcription factor, yet the authors claim MAPK is unchanged. Therefore, how does Elk1 phosphorylation decrease without upstream pathway suppression?
• The study does not investigate CREB, RSK, CaMK, or other signaling nodes upstream of Elk1, even though they are mentioned in the Discussion.

We appreciate your important suggestions. We verified the effect of surfactin on RANKL-induced CREB phosphorylation by Western blot analysis, but no modification activity was confirmed. Based on these results, we are considering the possibility of a direct action on the Elk1 molecule as a candidate mechanism for the negative regulation of osteoclast formation by surfactin. So, we added Supplemental Figure 3 and modified the Discussion in the revised manuscript (lines 341- 345).

 

2. The Discussion states that the surfactin used contains multiple analogs with different fatty-acid chain lengths and branching patterns, which may explain discrepancies with prior studies. However, the authors did not show any comparison with single-analog surfactin. As well as no examination of dose–response for individual analog classes.

Thank you for your important suggestions. As the reviewers pointed out, the impact of differences in surfactin composition on biological activity is an important topic for future research. However, we did not mention the structure or purity of the surfactin we used in this study. Therefore, we added this information and revised the Discussion (lines 355-359) and Materials Methods (line 84) sections of the manuscript with some references (38 and 39).

 

3. The manuscript reports NFκB and MAPK signaling were analyzed at a single time point (15 min). However, RANKL signaling is dynamic, and some regulators show biphasic responses.

We appreciate your critical suggestions. Preliminary experiments revealed that degradation of IκBα protein and MAPK phosphorylation are transiently induced in RAW264.7 cells 15 minutes after RANKL stimulation alone. This study focused on the effect of surfactin on the initial induction of NFATc1 expression by RANKL as a molecular mechanism for its negative regulation of osteoclast differentiation. Therefore, we examined this at a single optimized time point. To analyze the detailed molecular mechanism, we believe it will be necessary to investigate the temporal effects on these intracellular signals in future studies. However, we did not mention about it. So, we added Supplemental Figure 1 and 2 and modified the in Results (lines 229-231 and lines 246-249) and Discussions (lines 376-378) in the revised manuscript.

 

4. The author should measure the phospho and total NFκB (p65) levels in both cytoplasm and nucleus to determine the activation of NFκB. Additionally, the Western blot images (Figures 4-6) lack quantification.

Thank you for your suggestions. In this study, we monitored protein degradation IκBα and nuclear translocation of p65 as indicators of NF-κB pathway activation. However, we did not perform quantitative analysis of the blots. For the quantification of proteins, we performed the densitometric analyses on all the blots. We provided bar graphs and edited the Materials and Methods (lines 166-167), Figure 4-6 (new Figure 5, 6 and 7) in the revised manuscript. 

 

Minor Concerns

1. TRAP and F-Actin Images Need Higher Resolution

As noted by the reviewer, we have upgraded the TRAP and F-actin images to higher quality (Figure 2a and 2d).

 

2. The manuscript does not specify whether normality tests were performed. The sample size (n) is not stated in the figures. Whether experiments were repeated independently is unclear.

Thank you for your comments. We added the sample size and the statistical analysis method used to the Figure legends in the revised manuscript.

 

3. Some sentences in the Introduction and Discussion are overly long and should be streamlined.

According to the reviewer’s suggestion, we have made the Introduction and Discussion sections of the manuscript more concise.

Reviewer 2 Report

Comments and Suggestions for Authors

1. In Figures 4–6, surfactin alone appears to increase the phosphorylation or nuclear accumulation of several signaling molecules (p65, MAPKs, p–c-Jun, p–c-Fos, p–Elk1). However, in Figure 3, surfactin alone has little to no effect on the mRNA expression of Nfatc1, Acp5, Cathepsin K, and Oc-stamp. The authors conclude that surfactin does not affect RANKL-induced NF-κB/MAPK activation, but they do not comment on the apparent activation by surfactin alone.

2. The authors conclude that surfactin inhibits Elk1 phosphorylation independently of MAPK, but this claim is not experimentally demonstrated. Elk1 is classically phosphorylated by ERK and JNK. The authors show only one early time point (15 min for MAPK, 60 min for Elk1). It is possible that surfactin affects MAPK at different time points, different isoforms, or in a transient manner not captured in the experiments. Thus, claiming “MAPK-independent mechanism” is scientifically premature.

3. TRAP staining and F-actin images are presented, but quantification is incomplete. Missing: Mean nuclei per osteoclast, F-actin ring area/number quantification, Total cell number control (to rule out mild cytotoxicity), Representative high-magnification insets for ring structure quality.

4. Oc-stamp results contradict “late fusion inhibition” without mechanistic explanation. The authors state that surfactin may inhibit late-stage cell fusion, yet Oc-stamp mRNA is unchanged (Fig. 3). This contradicts the fusion-inhibition hypothesis unless a membrane-level biophysical mechanism is proven.

5. RAW264.7 is a convenient but non-ideal model for osteoclastogenesis: It bypasses M-CSF signaling. It responds differently than primary bone marrow macrophages. Many journals require primary cell validation for mechanistic claims.

6. No verification of surfactin purity or analog composition effects. The authors mention the mixture of analogs but do not provide: LC–MS data for batch composition, Purity %, Identity verification. Given their argument that analog differences explain discrepancies with Kuang et al., analytical confirmation is necessary.

minor concerns
1. The authors only mention ANOVA + post-hoc tests, but do not specify: Which figures used Dunnett vs. Tukey, Exact sample size (n) for each experiment, Whether normality/homogeneity tests were conducted, Whether blinding or randomization was used for cell counting.
2. Table 1 shows Oc-stamp reverse primer duplicated from Acp5 reverse primer (identical sequence!)—likely an error.
3. Figure numbering in text sometimes mismatches order.
4. Minor spacing and grammar errors throughout.
5. “induced (Figure 6A)” missing a word.

Author Response

Major Concerns

1. In Figures 4–6, surfactin alone appears to increase the phosphorylation or nuclear accumulation of several signaling molecules (p65, MAPKs, p–c-Jun, p–c-Fos, p–Elk1). However, in Figure 3, surfactin alone has little to no effect on the mRNA expression of Nfatc1, Acp5, Cathepsin K, and Oc-stamp. The authors conclude that surfactin does not affect RANKL-induced NF-κB/MAPK activation, but they do not comment on the apparent activation by surfactin alone.

Thank you for your valuable suggestions. As noted by reviewers, stimulation of surfactin alone activated NF-κB and MAPK-mediated signaling pathways. However, we do not mention about it. So, we added new reference and modified Discussion in the revised manuscript (lines 320-326 ).

 

2. The authors conclude that surfactin inhibits Elk1 phosphorylation independently of MAPK, but this claim is not experimentally demonstrated. Elk1 is classically phosphorylated by ERK and JNK. The authors show only one early time point (15 min for MAPK, 60 min for Elk1). It is possible that surfactin affects MAPK at different time points, different isoforms, or in a transient manner not captured in the experiments. Thus, claiming “MAPK-independent mechanism” is scientifically premature.

We appreciate your critical suggestions. Preliminary experiments revealed that degradation of IκBα protein and MAPK phosphorylation are transiently induced in RAW264.7 cells 15 minutes after RANKL stimulation alone. This study focused on the effect of surfactin on the initial induction of NFATc1 expression by RANKL as a molecular mechanism for its negative regulation of osteoclast differentiation. Therefore, we examined this at a single optimized time point. To analyze the detailed molecular mechanism, we believe it will be necessary to investigate the temporal effects on these intracellular signals in future studies. However, we did not mention about it. So, we added Supplemental Figure 1 and 2, and modified the in Results (lines 229-231 and lines 246-249) and Discussions (lines 376-378) in the revised manuscript.

 

3. TRAP staining and F-actin images are presented, but quantification is incomplete. Missing: Mean nuclei per osteoclast, F-actin ring area/number quantification, Total cell number control (to rule out mild cytotoxicity), Representative high-magnification insets for ring structure quality.

As noted by the reviewer, we have upgraded the TRAP and F-actin images to higher quality (Figure 2a and 2d). Additionally, we have added quantitative analysis of TRAP-positive cells (Figure 2c) and F-actin-positive cells (Figure 2e) in the revise manuscript.

 

4. Oc-stamp results contradict “late fusion inhibition” without mechanistic explanation. The authors state that surfactin may inhibit late-stage cell fusion, yet Oc-stamp mRNA is unchanged (Fig. 3). This contradicts the fusion-inhibition hypothesis unless a membrane-level biophysical mechanism is proven.

Thank you for your critical suggestions. Elucidation the molecular mechanisms by which surfactin affects osteoclast fusion requires multifaceted investigation, including the dynamics of various associated molecules and phospholipids. However, we did not mention about it. So, we modified the Discussions (lines 368-371) in the revised manuscript with some references (44-46)

 

5. 7 is a convenient but non-ideal model for osteoclastogenesis: It bypasses M-CSF signaling. It responds differently than primary bone marrow macrophages. Many journals require primary cell validation for mechanistic claims.

We appreciate your critical suggestions. According to the reviewer’s comments, we conducted experiments using bone marrow cells isolated from mouse tibiae and femurs and found that surfactin inhibits osteoclast differentiation. Therefore, we added new Figure 3 and modified Materials and Methods (lines 120-129), Results (lines 193-196), and Discussion (lines 292-294) in the revised manuscript.

 

6. No verification of surfactin purity or analog composition effects. The authors mention the mixture of analogs but do not provide: LC–MS data for batch composition, Purity %, Identity verification. Given their argument that analog differences explain discrepancies with Kuang et al., analytical confirmation is necessary.

Thank you for your important suggestions. As the reviewers pointed out, the impact of differences in surfactin composition on biological activity is an important topic for future research. However, we did not mention the structure or purity of the surfactin we used in this study. Therefore, we added this information and revised the Discussion (lines 355-359) and Materials Methods (line 84) sections of the manuscript with some references (38 and 39).

 

 

Minor Concerns

1. The authors only mention ANOVA + post-hoc tests, but do not specify: Which figures used Dunnett vs. Tukey, Exact sample size (n) for each experiment, whether normality/homogeneity tests were conducted, whether blinding or randomization was used for cell counting.

Thank you for your comments. We added the sample size and the statistical analysis method used to the Figure legends in the revised manuscript.

 

2. Table 1 shows Oc-stamp reverse primer duplicated from Acp5 reverse primer (identical sequence!)—likely an error.

We apologize for providing incorrect information. We have corrected the reverse primer sequence for Oc-stamp in Table 1.

 

3. Figure numbering in text sometimes mismatches order.

As noted by the reviewer, we checked the figure numbering and corrected it to match the order in the main manuscript.

 

4. Minor spacing and grammar errors throughout.

We carefully checked the manuscript and corrected spacing and grammatical errors.

 

5. “induced (Figure 6A)” missing a word.

Thank you for your suggestions. We have removed the “induced” and revised the manuscript (lines 263-265).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all of my concerns with the previous version of the manuscript. Therefore, I recommend the acceptance of this revised version of the manuscript.

Author Response

We would like to express our gratitude for the peer review of our research and for your valuable comments and suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has substantially improved; however, the description of the Elk1 regulatory mechanism remains overly definitive. Specifically, statements referring to a “MAPK-independent” inhibition of Elk1 (e.g., in the Discussion section and corresponding conclusions/figure schematic) are not fully supported by the experimental design, which evaluates MAPK activity at limited time points.

To accurately reflect the data presented, I request that the term “MAPK-independent” be replaced with more precise wording such as “not associated with detectable MAPK modulation at the examined time points.” This revision should be applied consistently wherever the MAPK-independent mechanism is stated.

Author Response

We appreciate your important suggestions. As you pointed out, this study lacks sufficient evidence to conclude that the effect of surfactin on osteoclast differentiation is induced in a “MAPK-independent” manner. Therefore, we modified the Discussions (lines 330-333 and 338-339) in the revised manuscript.