The Anti-Inflammatory Role of GPNMB in Post-Traumatic Osteoarthritis

Reviewer #1 (Public review):

Summary:

While previous studies by this group and others have demonstrated the anti-inflammatory properties of osteoactivin, its specific role in cartilage homeostasis and disease pathogenesis remains unknown.

Strengths:

Strengths of the study include its clinical relevance, given the lack of curative treatments for osteoarthritis, as well as the clarity of the narrative and the quality of most results."

Weaknesses:

A limitation of the study is the reliance on standard techniques; however, this is a minor concern that does not diminish the overall impact or significance of the work.

Comments on revisions:

The authors have satisfactorily addressed my concerns.

Reviewer #2 (Public review):

Summary:

This manuscript presents compelling evidence for a novel anti-inflammatory function of glycoprotein non-metastatic melanoma protein B (GPNMB) in chondrocyte biology and osteoarthritis (OA) pathology. Through a combination of in vitro, ex vivo, and in vivo models, including the destabilization of the medial meniscus (DMM) surgery in mice, the authors demonstrate that GPNMB expression is upregulated in OA-affected cartilage and that recombinant GPNMB treatment reduces the expression of key catabolic markers (MMPs, Adamts-4, and IL-6) without impairing anabolic gene expression. Notably, DBA/2J mice lacking functional GPNMB exhibit exacerbated cartilage degradation post-injury. Mechanistically, GPNMB appears to mitigate inflammation via the MAPK/ERK pathway. Overall, the work is thorough, methodologically sound, and significantly advances our understanding of GPNMB as a protective modulator in osteoarthritic joint disease. The findings could open pathways for therapeutic development.

Strengths:

(1) Clear hypothesis addressing a well-defined knowledge gap.

(2) Robust and multi-modal experimental design: includes human, mouse, cell-line, explant, and surgical OA models.

(3) Elegant use of DBA/2J GPNMB-deficient mice to mimic endogenous loss-of-function.

(4) Mechanistic insight provided through MAPK signaling analysis.

(5) Statistical analysis appears rigorous and the figures are informative.

Weaknesses:

(1) Clarify the strain background of the DBA/2J GPNMB+ mice: While DBA/2J GPNMB+ is described as a control, it would help to explicitly state whether these are transgenically rescued mice or another background strain. Are they littermates, congenic, or a separate colony?

(2) Provide exact sample sizes and variance in all figure legends: Some figures (e.g., Figure 2 panels) do not consistently mention how many replicates were used (biological vs. technical) for each experimental group. Standardizing this across all panels would improve reproducibility.

(3) Expand on potential sex differences: The DMM model is applied only in male mice, which is noted in the methods. It would be helpful if the authors added 1-2 lines in the discussion acknowledging potential sex-based differences in OA progression and GPNMB function.

(4) Visual clarity in schematic (Figure 7): The proposed mechanism is helpful but the text within the schematic is somewhat dense and could be made more readable with spacing or enlarged font. Also, label the MAPK/ERK pathway explicitly in panel B.

Comments on revisions:

The authors have addressed all the concerns raised in the initial review.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Reviews):

Weaknesses:

A limitation of the study is the reliance on standard techniques; however, this is a minor concern that does not diminish the overall impact or significance of the work.

We agree that standard techniques were utilized. We believe this approach enhances the reliability and reproducibility of our findings. These methods are well-validated in the field and allow for robust interpretation of the results presented.

Reviewer #2 (Public Reviews):

Weaknesses:

(1) Clarify the strain background of the DBA/2J GPNMB+ mice: While DBA/2J GPNMB+ is described as a control, it would help to explicitly state whether these are transgenically rescued mice or another background strain. Are they littermates, congenic, or a separate colony?

The following language was added to the manuscript, “The DBA/2J GPNMB+ mice are a coisogenic strain purchased from Jackson Laboratories. Jackon Laboratories generated these mice by knocking in the wild-type allele of Gpnmb into the DBA/2J background. By doing so, they rescued the phenotype of the DBA/2J mice. This description has been highlighted in our previous publications (Abdelmagid et al., 2014; Abdelmagid et al., 2015).”

(2) Provide exact sample sizes and variance in all figure legends: Some figures (e.g., Figure 2 panels) do not consistently mention how many replicates were used (biological vs. technical) for each experimental group. Standardizing this across all panels would improve reproducibility.

The manuscript has been updated to include replicates in each figure legend.

(3) Expand on potential sex differences: The DMM model is applied only in male mice, which is noted in the methods. It would be helpful if the authors added 1-2 lines in the discussion acknowledging potential sex-based differences in OA progression and GPNMB function.

To our knowledge there are no sexbased differences in OA progression and GPNMB function in the literature. It was initially reported that only male C57BL/6J mice (Jackson Laboratories) develop OA following DMM however, recent literature has shown that both male and female mice develop the disease (Hwang et al., 2021; Ma et al., 2007). For the purpose of this manuscript, only male mice were used to provide preliminary results, however, we plan to repeat the included studies in female mice in the near future.

(4) Visual clarity in schematic (Figure 7): The proposed mechanism is helpful, but the text within the schematic is somewhat dense and could be made more readable with spacing or enlarged font. Also, label the MAPK/ERK pathway explicitly in panel B.

We updated the schematic diagram in figure 7 and the figure legend.

Reviewer #1 (Recommendations for the Authors):

Several concerns must be addressed to improve the clarity and scientific rigor of the manuscript:

(1) Abstract: Specify which MMPs and MAPKs are modulated by osteoactivin.

We specified the MMPs and clarified that GPNMB plays a role in pERK inhibition following inflammation induced by IL-1β stimulation.

(2) Human explant validation: The regulation of MMP-9, MMP-13, and IL-6 should be validated in the human cartilage explant model to support the claim that "GPNMB has an anti-inflammatory role in human primary chondrocytes" (line 123). Additionally, the anatomical origin of the explants must be stated.

Thank you very much for the recommendation. We agree that validating the explant culture for MMP-9, MMP-13, and IL-6 would strengthen our data. Unfortunately, this experiment has been terminated and we no longer have access to the tissue. Human explants were obtained from discarded knee articular cartilage following arthroplasty. The manuscript has been updated to include this information.

(3) DBA/2J GPNMB expression: GPNMB is known to be produced as a truncated protein in DBA/2J cells. The manuscript should address why its expression is reduced. Does this involve mRNA instability? Also, the nomenclature "DBA/2J GPNMB+" versus "DBA/2J" is confusing, especially since both mRNA and protein are still detectable, albeit at reduced levels. Figure 2C is not convincing; therefore, Figures 2C and 2D can be omitted.

The following language was added to the manuscript, “Our results are consistent with the literature which shows that that the GPNMB gene in DBA/2J mice carries a nonsense mutation that leads to reduced RNA stability (Anderson et al., 2008).” We can appreciate that the nomenclature "DBA/2J GPNMB+" versus "DBA/2J" could be confusing. However, this is the standard language used in multiple publications, and we want to remain consistent with the literature. Based on your recommendation we have removed Figure 2 C and D and updated the methods and results sections accordingly.

(4) Figures 2J-L: The claim that gene expression changes are "significantly higher in DBA/2J animals compared to fold changes seen in chondrocytes from DBA/2J GPNMB+ controls" is not supported by the current presentation. The data should be plotted on the same graphs, and appropriate statistical analysis (e.g., two-way ANOVA) must be performed.

Graphs for figure 2 have been updated and the appropriate analyses have been performed.

(5) Figure 6: The GPNMB expression data in the presence and absence of IL-1β at 0 and 10 minutes are missing.

We apologize for the confusion. We corrected the mistake and removed the mention of the timepoints 0 and 10 minutes.

Reviewer #2 (Recommendations for the Authors):

Consider unifying terminology around "GPNMB" and "osteoactivin": The term "osteoactivin" is used in some contexts and "GPNMB" in others. Since the focus is GPNMB's role in cartilage, suggest using a single term throughout to prevent confusion.

Thank you for your comment. We include osteoactivin for clarification purposes once in the abstract, introduction and discussion.

In summary, we believe we have addressed all comments/concerns raised by the reviewers. We appreciate the opportunity to improve the quality of our manuscript.

References

Abdelmagid, S. M., Belcher, J. Y., Moussa, F. M., Lababidi, S. L., Sondag, G. R., Novak, K. M., Sanyurah, A. S., Frara, N. A., Razmpour, R., & Del Carpio-Cano, F. E. (2014). Mutation in osteoactivin decreases bone formation in vivo and osteoblast differentiation in vitro. The American journal of pathology, 184(3), 697-713.

Abdelmagid, S. M., Sondag, G. R., Moussa, F. M., Belcher, J. Y., Yu, B., Stinnett, H., Novak, K., Mbimba, T., Khol, M., Hankenson, K. D., Malcuit, C., & Safadi, F. F. (2015). Mutation in Osteoactivin Promotes Receptor Activator of NFκB Ligand (RANKL)-mediated Osteoclast Differentiation and Survival but Inhibits Osteoclast Function. J Biol Chem, 290(33), 2012820146. https://doi.org/10.1074/jbc.M114.624270

Anderson, M. G., Nair, K. S., Amonoo, L. A., Mehalow, A., Trantow, C. M., Masli, S., & John, S. W. (2008). GpnmbR 150Xallele must be present in bone marrow derived cells to mediate DBA/2J glaucoma. BMC genetics, 9(1), 1-14.

Hwang, H., Park, I., Hong, J., Kim, J., & Kim, H. (2021). Comparison of joint degeneration and pain in male and female mice in DMM model of osteoarthritis. Osteoarthritis and Cartilage, 29(5), 728738.

Ma, H.-L., Blanchet, T., Peluso, D., Hopkins, B., Morris, E., & Glasson, S. (2007). Osteoarthritis severity is sex dependent in a surgical mouse model. Osteoarthritis and Cartilage, 15(6), 695-700.