Decreased bone resorption in Ezh2 myeloid cell conditional knockout mouse model

Authors: CAVINESS, PERRY - Arkansas Children'S Nutrition Research Center (ACNC); DONGZHENG, GAI - University Arkansas For Medical Sciences (UAMS); LAZARENKO, OXANA - Arkansas Children'S Nutrition Research Center (ACNC); BLACKBURN, MICHAEL - Arkansas Children'S Nutrition Research Center (ACNC); FENGHUANG, ZHAN - University Arkansas For Medical Sciences (UAMS); JIN-RAN, CHEN - Arkansas Children'S Nutrition Research Center (ACNC)

Submitted to: Federation of American Societies for Experimental Biology Conference
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2023
Publication Date: 6/5/2023
Citation: Caviness, P.C., Dongzheng, G., Lazarenko, O.P., Blackburn, M.L., Fenghuang, Z., Jin-Ran, C. 2023. Decreased bone resorption in Ezh2 myeloid cell conditional knockout mouse model. Federation of American Societies for Experimental Biology Conference. 37(7):e23019. https://doi.org/10.1096/fj.202201673RR.
DOI: https://doi.org/10.1096/fj.202201673RR

Interpretive Summary: Osteoclastogenesis is the process by which new bone resorbing osteoclast cells are formed from existing stem cell precursors. Increased osteoclastogenesis can results in disorders associated with increased bone resorption such as osteoporosis. As such, identifying novel options for regulation new osteoclast formation is an important area of research. The polycomb group (PcG) protein enhancer of zeste homologue 2 (Ezh2), is associated with the regulation of numerous cellular processes and directly modifies histones, proteins around which DNA is wrapped to suppress expression of key genes. However, Ezh2's involvement in bone cell development is currently unknown. Here, a mouse breeding strategy was used to delete Ezh2 in osteoclast precursor cells. Deletions of Ezh2 in precursor cells led to a significant increase mouse bone mass in the first 6 months of life for both male and female mice. For male mice, bone mass was identical for mice with deletion of Ezh2 in just one chromosome (partial knockout) to those with Ezh2 deleted in both chromosomes (full knockout). For female mice, only the full knockout of Ezh2 led to optimal bone mass. Significantly less mature osteoclasts were found in the bone marrow of Ezh2 deletion mice compared to the controls. In addition, the number of inflammatory cytokines (which promote osteoclastogenesis) was lowered in bone tissue from Ezh2 deletion mice compared to controls. The deletion of Ezh2 in osteoclast precursors also led to an increase in the number of bone depositing osteoblasts present in bone tissue as well as an increase in the expression of osteoclastogenesis suppressing genes IRF8, MafB and Arg1. These finding lead us to believe that suppressing the expression of Ezh2 is a viable strategy to combat bone resorptive disorders.

Technical Abstract: Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis is important for developing novel treatments for osteoporosis and other disorders associated with bone deterioration as well as promoting healthy bone formation. The polycomb group (PcG) protein enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase is associated with epigenetic regulation of numerous cellular processes, but its involvement in bone cell development and homeostasis is not yet clear. Here, LysM-Cre mice were crossed with Ezh2flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional knockout of Ezh2 (CKO) in myeloid cell line resulted in significant increases in postnatal bone growth in the first 6 months of life for both male and female mice. For female mice, optimal bone mass was seen for mice with Ezh2 deleted in both chromosomes in a pair (f/f Cre+; CKO). For male mice, optimal bone mass was found after deletion of Ezh2 from just one chromosome (f/- Cre+) with no difference in bone phenotype between f/- Cre+ and CKO male mice. In addition to the gender specific difference in bone phenotype, Ezh2 CKO mice had significantly less macrophages (CD11b+) present in the bone marrow compared to control mice as well as significantly more mature osteoblasts and bone formation biomarkers present (P1NP, osteocalcin). Inflammatory array for protein lysed from bone tissue revealed deletion of Ezh2 decreased inflammatory milieu in both male and female mice compared to controls. Unexpectedly, myeloid cell deletion of Ezh2 also increased the number of mature osteoblast present in the bone. Deletion of Ezh2 also led to an increase in gene expression of osteoclast suppressive genes IRF8, MafB and Arg1 due to a decrease in the presence of the suppressive H3K27me3 epigenetic mark. These findings suggest that manipulation of Ezh2 expression may be a viable strategy to combat bone resorptive disorders such as osteoporosis or arthritis.