The anthocyanin delphinidin is a natural compound found as water‐soluble pigment in coloured fruits and berries. Anthocyanin‐rich diets have been proposed to have bone protective effects in humans and mice, but the underlying mechanisms remain unclear. In this study, we used a medaka (Oryzias latipes) osteoporosis model to test the effects of delphinidin on bone cells in vivo. In this model, inducible transgenic expression of receptor‐activator of NF‐kβ ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, similar to the situation in human osteoporosis patients. Using live imaging in medaka bone reporter lines, we show that delphinidin significantly reduces the number of osteoclasts after Rankl induction and protects bone integrity in a dose‐dependent manner. Our in vivo findings suggest that delphinidin primarily affects the de novo differentiation of macrophages into osteoclasts rather than the recruitment of macrophages to sites of bone resorption. For already existing osteoclasts, delphinidin treatment affected their morphology, leading to fewer protrusions and a more spherical shape. Apoptosis rates were not increased by delphinidin, suggesting that osteoclast numbers were reduced primarily by impaired differentiation from macrophage progenitors and reduced maintenance of pre‐existing osteoclasts. Importantly, and in contrast to previously reported cell culture experiments, no effect of delphinidin on osteoblast differentiation and distribution was observed in medaka in vivo. Our study is the first report on the effects of delphinidin on bone cells in fish embryos, which are a unique model system for compound testing that is suitable for live imaging of bone cell behaviour in vivo.

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The Bcl proteins play a critical role in apoptosis, as mutations in family members interfere with normal programmed cell death. Such events can cause cell transformation, potentially leading to cancer. Recent discoveries indicate that some viral proteins interfere with Bcl proteins either directly or indirectly; however, these data have not been systematically described. Some viruses encode proteins that reprogramme host cellular signalling pathways controlling cell differentiation, proliferation, genomic integrity, cell death, and immune system recognition. This review analyses and summarises the existing data and discusses how viral proteins interfere with normal pro- and anti-apoptotic functions of Bcl-2 and Bcl-xL. Particularly, this article focuses on how viral proteins, such as Herpesviruses, HTLV-1, HPV and HCV, block apoptosis and how accumulation of such interference predisposes cancer development. Finally, we discuss possible ways to prevent and treat cancers using a combination of traditional therapies and antiviral preparations that are effective against these viruses.

In mammals, osteoclasts differentiate from macrophages in the monocyte lineage. Although many factors driving osteoclast formation are known, the detailed processes underlying precursor recruitment, differentiation, and interaction of macrophages with other cell types involved in bone remodeling are poorly understood. Using live imaging in a transgenic medaka osteoporosis model, where ectopic osteoclasts are induced by RANKL expression, we show that a subset of macrophages is recruited to bone matrix to physically interact with bone-forming osteoblast progenitors. These macrophages subsequently differentiate into cathepsin K-(ctsk-) positive osteoclasts. One day later, other macrophages are recruited to clear dying osteoclasts from resorbed bone by phagocytosis. To better understand the molecular changes underlying these dynamic processes, we performed transcriptome profiling of activated macrophages upon RANKL induction. This revealed an upregulation of several bone-related transcripts. Besides osteoclast markers, we unexpectedly also found expression of osteoblast-promoting signals in activated macrophages, suggesting a possible non-cell autonomous role in osteogenesis. Finally, we show that macrophage differentiation into osteoclasts is dependent on inflammatory signals. Medaka deficient for TNFα or treated with the TNFα-inhibitor pentoxifylline exhibited impaired macrophage recruitment and osteoclast differentiation. These results show the involvement of inflammatory signals and the dynamics of a distinct subset of macrophages during osteoclast formation.

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Matrix metalloproteases (MMPs) play crucial roles in extracellular matrix (ECM) modulation during osteoclast-driven bone remodeling. In the present study, we used transcriptome profiling of bone cells in a medaka model for osteoporosis and bone regeneration to identify factors critical for bone remodeling and homeostasis. This identified mmp13b, which was strongly expressed in osteoblast progenitors and upregulated under osteoporotic conditions and during regeneration of bony fin rays. To characterize the role of mmp13b in bone remodeling, we generated medaka mmp13b mutants by CRISPR/Cas9. We found that mmp13b mutants form normal numbers of osteoblasts and osteoclasts. However, osteoclast activity was severely impaired under osteoporotic conditions. In mmp13b mutants and embryos treated with the MMP13 inhibitor CL-82198, unmineralized collagens and mineralized bone matrix failed to be degraded. In addition, the dynamic migratory behavior of activated osteoclasts was severely affected in mmp13b mutants. Expression analysis showed that maturation genes were downregulated in mmp13b deficient osteoclasts suggesting that they remain in an immature and non-activated state. We also found that fin regeneration was delayed in mmp13b mutants with a concomitant alteration of the ECM and reduced numbers of osteoblast progenitors in regenerating joint regions. Together, our findings suggest that osteoblast-derived Mmp13b alters the bone ECM to allow the maturation and activation of osteoclasts during bone remodeling in a paracrine manner. Mmp13b-induced ECM alterations are also required to facilitate osteoblast progenitor recruitment and full regeneration of bony fin rays.

Calcium phosphate (CaP) is the inorganic part of hard tissues, such as bone, teeth and tendons, and has a high biocompatibility and good biodegradability. Therefore, CaP nanoparticles functionalized with DNA encoding bone anabolic factors are promising carrier-systems for future therapeutic development. Here, we analysed CaP nanoparticles in a genetically modified medaka fish model, where osteoporosis-like lesions can be induced by transgenic expression of receptor activator of nuclear factor kappa-B ligand (Rankl). Rankl-transgenic medaka were used to visualize and understand effects of microinjected functionalized CaP nanoparticles during modulation of osteoclast activity in vivo. For this, we synthetized multi-shell CaP nanoparticles by rapid precipitation of calcium lactate and ammonium hydrogen phosphate followed by the addition of plasmid DNA encoding the osteoclastogenesis inhibitory factor osteoprotegerin-b (Opgb). An additional layer of poly(ethyleneimine) was added to enhance cellular uptake. Integrity of the synthesized nanoparticles was confirmed by dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Fluorescently labelled CaP nanoparticles were microinjected into the heart, trunk muscle or caudal fins of Rankl-transgenic medaka embryos that expressed fluorescent reporters in various bone cell types. Confocal time-lapse imaging revealed a uniform distribution of CaP nanoparticles in injected tissues and showed that nanoparticles were efficiently taken up by macrophages that subsequently differentiated into bone-resorbing osteoclasts. After Rankl induction, fish injected with Opg-functionalized nanoparticles showed delayed or absent degradation of mineralized matrix, i.e. a lower incidence of osteoporosis-like phenotypes. This is proof of principle that CaP nanoparticles can be used as carriers to efficiently deliver modulatory compounds to osteoclasts and block their activity.

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