Abstract
To identify the repertoire of ephrin genes that might regulate endochondral bone fracture repair, we examined changes in ephrin ligand and receptor (Eph) gene expression in fracture callus tissues during bone fracture healing. Ephrin and Eph proteins were then localized in the fracture callus tissues present when changes in gene expression were observed. Ephrin gene expression was widespread in fracture tissues, but the repertoire of ephrin genes with significant changes in expression that might suggest a regulatory role in fracture callus development was restricted to the ephrin A family members Epha4, Epha5 and the ephrin B family member Efnb1. After 3 weeks of healing, Epha4 fracture expression was downregulated from 1.3- to 0.8-fold and Epha5 fracture expression was upregulated from 1.2- to 1.5-fold of intact contralateral femur expression, respectively. Efnb1 expression was downregulated from 1.5- to 1.2-fold after 2 weeks post-fracture. These ephrin proteins were localized to fracture callus prehypertrophic chondrocytes and osteoblasts, as well as to the periosteum and fibrous tissues. The observed positive correlation between mRNA levels of EfnB1 with Col10 and Epha5 with Bglap, together with colocalized expression with their respective proteins, suggest that EfnB1 is a positive mediator of prehypertrophic chondrocyte development and that Epha5 contributes to osteoblast-mediated mineralization of fracture callus. In contrast, mRNA levels of Epha4 and Efnb1 correlated negatively with Bglap, thus suggesting a negative role for these two ephrin family members in mature osteoblast functions. Given the number of family members and widespread expression of the ephrins, a characterization of changes in ephrin gene expression provides a basis for identifying ephrin family members that might regulate the molecular pathways of bone fracture repair. This approach suggests that a highly restricted repertoire of ephrins, EfnB1 and EphA5, are the major mediators of fracture callus cartilage hypertrophy and ossification, respectively, and proposes candidates for additional functional study and eventual therapeutic application.
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Acknowledgements
The authors thank Nancy Lowen for expert assistance with the immunohistochemistry. This work was supported by Merit Review Award # 5 I01 BX002519-04 (WX, CHR) from the United States (US) Department of Veterans Affairs Biomedical Research and Development Program. SM is the recipient of a Senior Research Career Scientist Award from the U.S. Department of Veterans Affairs. CHR is a Research Biologist at the Jerry L. Pettis Memorial Veterans Administration Medical Center, Loma Linda, CA. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.
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Online Resource 3
. Bone formation marker protein isotype control immunohistochemistry. COLX and BGLAP IgG isotype controls in fractured femur tissues harvested at 3 weeks (a) and 4 weeks (b), respectively. Insets are low-magnification images of the section. f, fracture; ob, osteoblasts; ocy, osteocytes. Scale bars = 200 um. (TIF 4320 KB)
Online Resource 4
. Expression of ephrin A and ephrin B proteins in unfractured femur tissues. EFN and EPH expression is localized by immunohistochemistry in the contralateral femur from each of the fracture tissues in Fig 3. EFNA2 in the bone marrow at 2 weeks (a). EFNB1 in the periosteum at 3 weeks (b). EPHA4 in the bone marrow and in the distal articular chondrocytes (center inset) at 4 weeks (c). EFNB2 in the metaphysis at 1 week (d). EPHA5 in the bone marrow at 4 weeks (e). EPHB2 in the periosteum at 3 weeks (f). Lower left insets are low-magnification images of the section. Lower right insets show the distal growth plate. ac, articular cartilage; bm, bone marrow; cb, cortical bone; gpc, growth plate chondrocytes; mk, megakaryocyte; ob, osteoblasts; p, periosteum. Scale bars = 100 um. (TIF 7760 KB)
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. Ephrin isotype control immunohistochemistry. EFN and EPH IgG isotype controls in unfractured femur (a, c, e, f) and fractured femur (b, d, f, g) tissues harvested at 1 week (a, b), 2 weeks (c, d), 3 weeks (e, f) and 4 weeks (g, h). Insets are low-magnification images of the section. bm, bone marrow; f, fracture; mk, megakaryocyte; hc, hypertrophic chondrocytes; ob, osteoblasts; ocy, osteocytes; p, periosteum, phc, prehypertrophic chondrocytes. Scale bars = 100 um. (TIF 10268 KB)
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Kaur, A., Xing, W., Mohan, S. et al. Changes in ephrin gene expression during bone healing identify a restricted repertoire of ephrins mediating fracture repair. Histochem Cell Biol 151, 43–55 (2019). https://doi.org/10.1007/s00418-018-1712-7
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DOI: https://doi.org/10.1007/s00418-018-1712-7