osteocytes

Degradation-Resistant Hypoxia Inducible Factor-2α in Murine Osteocytes Promotes a High Bone Mass Phenotype

AUTHORS

Sarah V. Mendoza MS, Deepa K. Murugesh BS, Blaine A. Christiansen PhD, Zoe O. Genetos, Gabriela G. Loots PhD, Damian C. Genetos PhD, Clare E. Yellowley PhD

ABSTRACT

Molecular oxygen levels vary during development and disease. Adaptations to decreased oxygen bioavailability (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are composed of an oxygen-dependent α subunit (HIF-α), of which there are two transcriptionally active isoforms (HIF-1α and HIF-2α), and a constitutively expressed β subunit (HIFβ). Under normoxic conditions, HIF-α is hydroxylated via prolyl hydroxylase domain protein (PHD) and targeted for degradation via von-Hippel Lindau (VHL). Under hypoxic conditions, hydroxylation via PHD is inhibited, allowing for HIF-α stabilization and induction of target transcriptional changes. Our previous studies showed that Vhl deletion in osteocytes (Dmp1-cre; Vhlf/f) resulted in HIF-α stabilization and generation of a high bone mass (HBM) phenotype. The skeletal impact of HIF-1α accumulation has been well characterized, however, the unique skeletal impacts of HIF-2α remain understudied. Because osteocytes orchestrate skeletal development and homeostasis, we investigated the role of osteocytic HIF-α isoforms in driving high bone mass phenotypes via osteocyte-specific loss- and gain of function HIF-1α and HIF-2α mutations in C57BL/6 female mice. Deletion of Hif1a or Hif2a in osteocytes showed no effect on skeletal microarchitecture. Constitutively stable, degradation-resistant HIF-2α (HIF-2α cDR), but not HIF-1α cDR, generated dramatic increases in bone mass, enhanced osteoclast activity, and expansion of metaphyseal marrow stromal tissue at the expense of hematopoietic tissue. Our studies reveal a novel influence of osteocytic HIF-2α in driving high bone mass phenotypes that can potentially be harnessed pharmacologically to improve bone mass and reduce fracture risk.

Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection

AUTHORS

Tetsuya Yoshimoto, Mizuho Kittaka, Andrew Anh Phuong Doan, Rina Urata, Matthew Prideaux, Roxana E. Rojas, Clifford V. Harding, W. Henry Boom, Lynda F. Bonewald, Edward M. Greenfield & Yasuyoshi Ueki

ABSTRACT

The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.

Osteocyte CIITA aggravates osteolytic bone lesions in myeloma

AUTHORS

Huan Liu, Jin He, Rozita Bagheri-Yarmand, Zongwei Li, Rui Liu, Zhiming Wang, Duc-hiep Bach, Yung-hsing Huang, Pei Lin, Theresa A. Guise, Robert F. Gagel & Jing Yang

ABSTRACT

Osteolytic destruction is a hallmark of multiple myeloma, resulting from activation of osteoclast-mediated bone resorption and reduction of osteoblast-mediated bone formation. However, the molecular mechanisms underlying the differentiation and activity of osteoclasts and osteoblasts within a myelomatous microenvironment remain unclear. Here, we demonstrate that the osteocyte-expressed major histocompatibility complex class II transactivator (CIITA) contributes to myeloma-induced bone lesions. CIITA upregulates the secretion of osteolytic cytokines from osteocytes through acetylation at histone 3 lysine 14 in the promoter of TNFSF11 (encoding RANKL) and SOST (encoding sclerostin), leading to enhanced osteoclastogenesis and decreased osteoblastogenesis. In turn, myeloma cell–secreted 2-deoxy-D-ribose, the product of thymidine catalyzed by the function of thymidine phosphorylase, upregulates CIITA expression in osteocytes through the STAT1/IRF1 signaling pathway. Our work thus broadens the understanding of myeloma-induced osteolysis and indicates a potential strategy for disrupting tumor-osteocyte interaction to prevent or treat patients with myeloma bone disease.

Gene expression of intracortical bone demonstrates loading-induced increases in Wnt1 and Ngf and inhibition of bone remodeling processes

AUTHORS

Taylor L.Harris, Matthew J.Silva

ABSTRACT

Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.

Gene expression of intracortical bone demonstrates loading-induced increases in Wnt1 and Ngf and inhibition of bone remodeling processes

AUTHORS

Taylor L.Harris, Matthew J.Silva

ABSTRACT

Osteocytes are the primary mechanosensitive cells in bone. However, their location in mineralized matrix has limited the in vivo study of osteocytic genes induced by mechanical loading. Laser Capture Microdissection (LCM) allows isolation of intracortical bone (Intra-CB), enriched for osteocytes, from bone tissue for gene expression analysis. We used microarray to analyze gene expression from mouse tibial Intra-CB dissected using LCM 4 h after a single loading bout or after 5 days of loading. Osteocyte enrichment was supported by greater expression of Sost, Dmp1, Dkk1, and Mepe in Intra-CB regions vs. Mixed regions containing periosteum and muscle (fold-change (FC) = 3.4, 2.2, 5.1, 3.0, respectively). Over 150 differentially expressed genes (DEGs) due to loading (loaded vs. contralateral control) in Intra-CB were found on Day 1 and Day 5, but only 10 genes were differentially expressed on both days, including Ngf (Day 1 FC = 13.5, Day 5 FC = 11.1) and Wnt1 (Day 1 FC = 1.5, Day 5 FC = 5.1). The expression of Ngf and Wnt1 within Intra-CB was confirmed by in situ hybridization, and a significant increase in number of Wnt1 mRNA molecules occurred on day 1. We also found changes in extracellular matrix remodeling with Timp1 (FC = 3.1) increased on day 1 and MMP13 (FC = 0.3) decreased on day 5. Supporting this result, IHC for osteocytic MMP13 demonstrated a marginal decrease due to loading on day 5. Gene Ontology (GO) biological processes for loading DEGs indicated regulation of vasculature, neuronal and immune processes while cell-type specific gene lists suggested regulation of osteoclast, osteoblast, and endothelial related genes. In summary, microarray analysis of microdissected Intra-CB revealed differential regulation of Ngf, Wnt1, and MMP13 due to loading in osteocytes.