TAM receptors control actomyosin dynamics in osteoclasts via RHOA-COFILIN-MYOSIN II signaling

Why I highlight this work

This preprint investigates relevant pathways for cell migration, amoeboid movement, and osteoclast activation that are useful to address in treating bone metastasis in breast cancer. MerTk is a receptor that seems to regulate the conversion to activated osteoclasts and promote bone loss. The pharmacological inhibition of the cellular molecules RHOA-ROCK and COFILIN can be helpful to establish personalized therapies aiming to treat patients with metastases.
The discovery of the pathway by which osteoclasts’ activation can be modulated opens the opportunity for drug repositioning and the application of Cofilin inhibitory approaches to other bone diseases, such as rheumatoid arthritis,osteoporosis,s and periodontitis.

 

Created in BioRender. Carvalho, N. (2025) https://BioRender.com/n42k7pz

Background

TAM (Tyro-3, Axl, MerTk) receptors belong to the tyrosine kinase family and recognize apoptotic cells bound by soluble ligands, Protein S and Gas6, which induce phagocytosis of the target cells. This process of apoptotic cell engulfment is termed efferocytosis and is important to downregulate the inflammatory response during a tissue repair process. Although TAM receptors perform virtually the same function, their intracellular cascade may be different depending on the physiological context. Nevertheless, they are undoubtedly necessary for the cytoskeleton alterations that precede efferocytosis [1-3]. The role of these receptors in “classic” phagocytes, macrophages and dendritic cells is well known, but their function in specialized tissues is still less explored.

During breast cancer metastasis to the bone, the bone-resident myeloid cells, osteoclasts, perform an exacerbated bone resorption that is detrimental to the tissue structure. The TAM receptors Tyro-3 and MerTk impact the activation of osteoclasts and may be involved in disease progression [4-5]. Some therapies aim to inhibit osteoclast activation or migration, but with several side effects that make their clinical use harder.

This preprint shows how TAM receptors differentially regulate osteoclast migration through RHOA-COFILIN-MYOSIN II signaling, which are molecules involved in cell contraction and actin polymerization.

Key findings:

MerTk deficiency in myeloid cells reduces osteoclastogenesis

Figure 1 of the preprint shows that, by microcomputed tomography (µCT), the authors observed increased bone volume, trabecular number, and thickness. When MerTk was depleted in myeloid cells, they found a reduced number of osteoclasts within the tissue, suggesting the role of this receptor in bone remodeling.

During osteoclastogenesis, TAM receptors are downregulated

The molecule responsible for osteoclast activation, RANKL, also decreased TAM receptors’ expression. The authors measured mRNA from precursor cells activated by RANKL and found negligible levels of Tyro-3, Axl and MerTk gene expression. Furthermore, the precursor cells expressed a higher level of MerTk, whereas the mature osteoclasts had lower levels, showing that MerTk is downregulated during cell activation.

Osteoclast precursors’ amoeboid migration is regulated by MerTk and COFILIN phosphorylation

A shift from an amoeboid to a mesenchymal migration mode, corroborated by increased cell directionality, was seen in the experiments where MerTk was deleted (Figure 3 of the preprint). Since osteoclast migration into bone resorption sites is important for its role in homeostasis, the authors described how cell physiology can be affected by MerTk signaling.

Additionally, the observed increase of rounded cells was correlated with low actin polymerization, which is dependent on COFILIN phosphorylation. Therefore, the lack of MerTk led to COFILIN phosphorylation and decreased migration.

Tyro-3 inhibits RHOA-ROCK pathway to regulate cell migration

The regulator of actin polymerization is the small GTPase RHO, which is inhibited by the canonical TAM ligand, Protein S (Pros1). With or without MerTk expression, Pros1 induced COFILIN phosphorylation, something that did not happen when Tyro-3 was depleted. Interestingly, these results contrast with the existing literature on MerTk.

The Tyro-3 receptor was also activated by Pros1, so the experiment was repeated, and the authors found that RHOA is strongly activated in the absence of Tyro-3, which must be activated by MerTk. In summary, Pros1-mediated activation of MerTk leads to cell migration and polarity, but if Tyro-3 is engaged, this phenotype is reversed by ROCK inhibition.

MerTk or Tyro-3 activation impacts osteolytic bone disease associated with breast cancer bone metastases

In mice lacking MerTk, after injection of a cell lineage of breast cancer, there was increased bone volume, trabecular number, and trabecular thickness compared to MerTk-expressing individuals. This suggests that osteolysis in cancer may be mediated by MerTk. Also, Tyro-3 deletion led to the same phenotype, showing that in vivo, both receptors are important, corroborating the inhibitory function of MERTK as well as TYRO3 in osteoclasts.

Future directions and questions for the authors 

Major Questions

Q1: While the data from the in vitro experiments is satisfactory, the observed phenotype in the in vivo steps was ineffective, which can be explained by several factors, such as temperature and stress. It would be great if the authors could address how temperature affects the biology of TAM receptors and actin polymerization, since the literature demonstrates that mice kept at required sub-thermoneutral temperatures (~22°C) suffer from chronic stress [6].

Q2: Given that various anti-Axl medicines are available for cancer and are regarded as the most therapeutically advanced [7], could the authors please clarify why Tyro-3 and MerTk were chosen over Axl in the investigation of cell migration and cancer metastasis?

Given that MerTk exhibits affinity for Gas6, it would be beneficial for the authors to incorporate this ligand into the cell migration analysis too.

Q3: There is no description of the animal facility (diet, humidity, temperature), sample size calculation, or the procedures of breast cancer cell injection. Could this be included in the ‘Materials & Methods’ section?

Q4: Since the C57Bl6/J is a mouse lineage, perhaps the authors can describe which sub-lineage was used to perform the in vivo experiments [8]? Also, the sublineage of mice may explain the differences observed among the in vivo assays.

Q5: The sample effort of Figure 5 was remarkably lower than the other figures, which might introduce bias in the analysis. Therefore, in order to improve statistical reproducibility, the experiments could be repeated with more subjects.

Q6: The in vivo tests are inconclusive about the predominant function of Tyro-3 or MerTk in the phenotype since the differences between controls and knockout mice are statistically insignificant. The temperature of the animal housing can be a stressor that affects the experiment’s resolution [8]. What role do adrenaline and cortisol play in cell migration to bone?

Minor questions

Q1: For the study design, a scheme illustration would be really helpful to the reader.

Q2: On p.15, in the “Statistics and reproducibility” section, given that normal distribution of samples is a necessity for statistical parametric tests, the authors could apply the normal distribution test prior to analysis.

Q3: For Figures 2 and 3: To make these figures self-explanatory, the trajectory plots should include subtitles to identify what the red or black lines mean.

Disclaimer

Artificial intelligence tools (QuillBot) were used to assist in language editing and improve clarity during the revision of this manuscript.

References

[1] Miao, Y. R., Rankin, E. B., & Giaccia, A. J. (2024). Therapeutic targeting of the functionally elusive TAM receptor family. Nature Reviews Drug Discovery, 23(3), 201-217.

[2] Doran, A. C., Yurdagul Jr., A., & Tabas, I. (2020). Efferocytosis in health and disease. Nature Reviews Immunology, 20(4), 254-267.

[3] Boada-Romero, E., Martinez, J., Heckmann, B. L., & Green, D. R. (2020). The clearance of dead cells by efferocytosis. Nature Reviews Molecular Cell Biology, 21(7), 398-414.

[4] Rothlin, C. V., Leighton, J. A., & Ghosh, S. (2014). Tyro3, Axl, and Mertk receptor signaling in inflammatory bowel disease and colitis-associated cancer. Inflammatory bowel diseases, 20(8), 1472-1480.

[5] Song, X., Wei, C., & Li, X. (2022). The signaling pathways associated with breast cancer bone metastasis.  Frontiers in Oncology, 12, 855609.

[6] Metze, M. (2016). Thermal neutral zone technology: the doorway to better research results. Lab Animal, 45(7), 274.

[7] Yadav, M., Sharma, A., Patne, K., Tabasum, S., Suryavanshi, J., Rawat, L., … & Sabarwal, A. (2025). AXL signaling in cancer: from molecular insights to targeted therapies. Signal Transduction and Targeted Therapy, 10(1), 37.

[8] Antonio Enriquez, J. (2019). Mind your mouse strain. NATURE METABOLISM, 1(1), 5-7.

Tags: bone metastasis, osteoclast, tam receptor

doi: https://doi.org/10.1242/prelights.41631

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