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Summary Ionizing radiation exposure results in acute and delayed bone marrow suppression. Treatment of mice with 16,16-dimethyl prostaglandin E2 (dmPGE2) prior to lethal ionizing radiation (IR) facilitates survival, but the cellular and molecular mechanisms are unclear. In this study we show that dmPGE2 attenuates loss and enhances recovery of bone marrow cellularity, corresponding to a less severe hematopoietic stem cell nadir, and significantly preserves long-term repopulation capacity and progenitor cell function. Mechanistically, dmPGE2 suppressed hematopoietic stem cell (HSC) proliferation through 24 h post IR, which correlated with fewer DNA double-strand breaks and attenuation of apoptosis, mitochondrial compromise, oxidative stress, and senescence. RNA sequencing of HSCs at 1 h and 24 h post IR identified a predominant interference with IR-induced p53-downstream gene expression at 1 h, and confirmed the suppression of IR-induced cell-cycle genes at 24 h. These data identify mechanisms of dmPGE2 radioprotection and its potential role as a medical countermeasure against radiation exposure.

Background and aims Mutations in the 5'-nucleotidase ecto (NT5E) gene that encodes CD73, a nucleotidase that converts AMP to adenosine, are linked to arterial calcification. However, the role of purinergic receptor signaling in the pathology of intimal calcification is not well understood. In this study, we examined whether extracellular nucleotides acting via P2Y 2 receptor (P2Y 2 R) modulate arterial intimal calcification, a condition highly correlated with cardiovascular morbidity. Methods Apolipoprotein E, P2Y 2 R double knockout mice ( ApoE −/− P2Y 2 R −/− ) were used to determine the effect of P2Y 2 R deficiency on vascular calcification in vivo . Vascular smooth muscle cells (VSMC) isolated from P2Y 2 R −/− mice grown in high phosphate medium were used to assess the role of P2Y 2 R in the conversion of VSMC into osteoblasts. Luciferase-reporter assays were used to assess the effect of P2Y 2 R on the transcriptional activity of Runx2. Results P2Y 2 R deficiency in ApoE −/− mice caused extensive intimal calcification despite a significant reduction in atherosclerosis and macrophage plaque content. The ectoenzyme apyrase that degrades nucleoside di- and triphosphates accelerated high phosphate-induced calcium deposition in cultured VSMC. Expression of P2Y 2 R inhibits calcification in vitro inhibited the osteoblastic trans -differentiation of VSMC. Mechanistically, expression of P2Y 2 R inhibited Runx2 transcriptional activation of an osteocalcin promoter driven luciferase reporter gene. Conclusions This study reveals a role for vascular P2Y 2 R as an inhibitor of arterial intimal calcification and provides a new mechanistic insight into the regulation of the osteoblastic trans -differentiation of SMC through P2Y 2 R-mediated Runx2 antagonism. Given that calcification of atherosclerotic lesions is a significant clinical problem, activating P2Y 2 R may be an effective therapeutic approach for treatment or prevention of vascular calcification.

Networking between hematopoietic stem cells (HSC) and cells of the hematopoietic niche is not only critical for the maintenance of stem cell function, but also for the competence of the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). We previously documented that close interactions between OM, osteoblasts (OB) and megakaryocytes (MK) are critical for sustaining in vitro and in vivo stem and progenitor cell function. To understand the importance of OM-OB-MK interactions within the niche, we performed 3D tissue cytometry on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, OB, MK and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. Next, we performed single cell mRNAseq and CyTOF to assess genetic and proteomic expression changes in OM following their interaction with MK. These studies revealed the upregulation of CD166 and embigin on OM via MK interactions. We then examined how these molecules impact hematopoietic function in clonogenic assays. When these assays were initiated with CD166 KO OM or treated with anti-embigin blocking antibodies, we established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. Our data, for the first time, reveal intimate spatial interactions between OM, OB, MK and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, OB and MK and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function.

Bone metastases are incurable. The bone microenvironment has always been a suspect for this clinical enigma, but the exact mechanisms have been unclear. In this issue of Cancer Cell, Zheng and colleagues provide evidence that chemotherapy itself induces chemoresistance of bone metastases, mediated by osteoblast Jagged1-induced tumor Notch signaling.

Understanding the mechanism by which tumor cells influence osteoclast differentiation is crucial for improving treatment of osteolytic metastasis. Here, we report broad microRNA (miRNA) expression changes in differentiating osteoclasts after exposure to tumor-conditioned media, in part through activation of NFκB signaling by soluble intracellular adhesion molecule (sICAM1) secreted from bone-metastatic cancer cells. Ectopic expression of multiple miRNAs downregulated during osteoclastogenesis suppresses osteoclast differentiation by targeting important osteoclast genes. Intravenous delivery of these miRNAs in vivo inhibits osteoclast activity and reduces osteolytic bone metastasis. Importantly, serum levels of sICAM1 and two osteoclast miRNAs, miR-16 and miR-378, which are elevated in osteoclast differentiation, correlate with bone metastasis burden. These findings establish miRNAs as potential therapeutic targets and clinical biomarkers of bone metastasis.