Osteocyte Transcriptome Mapping Identifies a Molecular Landscape Controlling Skeletal Homeostasis and Susceptibility to Skeletal Disease

Abstract

Osteocytes are master regulators of the skeleton. We map the transcriptome of osteocytes at different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cell-network. We define an osteocyte transcriptome signature, 1239 genes that distinguishes osteocytes from other cells. 77% have no known role in the skeleton.We show they are enriched for genes controlling neuronal network formation, suggesting this program is important in the osteocyte network. We evaluated 19 skeletal parameters in 733 mouse lines with functional-gene-deletions and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human homologues that cause monogenic skeletal dysplasias (P=6x10 -17 ), and associated with polygenic diseases, osteoporosis (P=1.8×10 -13 ), and osteoarthritis (P=2.6×10 -6 ). This reveals the molecular landscape that regulates osteocyte network formation and function, and establishes the importance of osteocytes in human skeletal disease. IntroductionThe skeleton is a highly dynamic structure that changes in shape and composition throughout life. Osteocytes are the most abundant cell type in bone and have emerged as master regulators of the skeleton. These enigmatic cells are connected via ramifying dendritic processes that form a complex multicellular network distributed throughout mineralized bone 1,2 .The scale and complexity of the osteocyte network is comparable to neurons in the brain, with 42 billion osteocytes present in the human skeleton forming 23 trillion connections 2,3 . This network enables osteocytes to detect and respond to mechanical strain, hormones and local growth factors and cytokines 1 . The network responds by regulating the formation and activity of osteoclasts and osteoblasts, instructing these cells to repair damaged bone, controlling bone mass and composition, and ensuring the optimal distribution of bone tissue in response to mechanical stress. Osteocytes also remove and replace bone surrounding the osteocyte network by the process of perilacunar remodeling, liberating calcium and phosphate in response to endocrine demands 4 . These features allow the osteocyte network to maintain both the structural integrity of the skeleton and mineral homeostasis. Osteocytes also have regulatory functions beyond the skeleton, including in skeletal muscle, adipose tissue, the central nervous system and in the control of phosphate homeostasis and energy expenditure, indicating the network acts as an important and endocrine organ 5-8 .Although osteocytes are pivotal in controlling the skeleton, the molecular programs that regulate their formation and function are poorly defined. Osteocytes are entombed in bone making them challenging to isolate and study. As a result osteocytes have been omitted from large-scale efforts to map tissue-specific transcriptomes 9-12 and studies of their transcriptome are limited [13][14][15][16] . Consequently, the influenc...

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption

“…This may suggest that while osteomorphs do not express TRAP protein at baseline, they are poised to express the protein when they are required to resorb bone again. This separation between Acp5 gene and TRAP protein expression has also been described in osteocytes ( Youlten et al., 2020 ). Regardless, osteoclast recycling provides a mechanism to explain the recent emergence of a rebound phenomenon, in which some patients have been reported to develop accelerated bone loss and rebound vertebral fractures 3–16 months after cessation of anti-RANKL treatment ( Anastasilakis et al., 2017a ; 2017b ; McClung et al., 2017 ; Popp et al., 2016 ; Tsourdi et al., 2017 ; Zanchetta et al., 2018 ).…”

Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption

“…This may suggest that while osteomorphs do not express TRAP protein at baseline, they are poised to express the protein when they are required to resorb bone again. This separation between Acp5 gene and TRAP protein expression has also been described in osteocytes (Youlten et al, 2020). Regardless, osteoclast recycling provides a mechanism to explain the recent emergence of a rebound phenomenon, in which some patients have been reported to develop accelerated bone loss and rebound vertebral fractures 3-16 months after cessation of anti-RANKL treatment (Anastasilakis et al, 2017a;2017b;McClung et al, 2017;Popp et al, 2016;Tsourdi et al, 2017;Zanchetta et al, 2018).…”

Opportunities and Challenges in Functional Genomics Research in Osteoporosis: Report From a Workshop Held by the Causes Working Group of the Osteoporosis and Bone Research Academy of the Royal Osteoporosis Society on October 5th 2020

“…Since bone-specific pathways are thought most likely to underlie skeletal phenotypes, if a gene is found to be expressed in bone, this is assumed to increase the likelihood that it underlies a given osteoporosis genetic association signal. This approach has been facilitated by description of the “osteocyte signature” ( 47 ), referring to the set of genes expressed preferentially in osteocytes, which was used to interrogate the genetic signals identified by Morris et al ( 22 ). As described below, information from skeletal phenotyping of mouse lines can also help to identify genes which are likely to underlie genetic association signals relevant to osteoporosis, as are previous reports that the gene in question is related to a skeletal disorder in humans.…”

“…About 50% of mice with outlier phenotypes have deletions of genes that have not been functionally annotated to the skeleton and are not known to be related to human skeletal disease. Integration of large scale mouse phenotype data with GWAS ( 20 , 22 ) and other cross-species multi-”omic” datasets ( 47 ) thus provide a rich resource to identify new genes and mechanisms involved in the pathogenesis of osteoporosis and monogenic human skeletal disorders ( 65 , 68 , 69 ).…”