Yayoi Izu

Osteoclast-specific Dicer gene deficiency suppresses osteoclastic bone resorption.

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010.

Angiotensin II Type 2 Receptor Blockade Increases Bone Mass

Renin angiotensin system (RAS) regulates circulating blood volume and blood pressure systemically, whereas RAS also plays a role in the local milieu. Previous in vitro studies suggested that RAS may be involved in the regulation of bone cells. However, it was not known whether molecules involved in RAS are present in bone in vivo. In this study, we examined the presence of RAS components in adult bone and the effects of angiotensin II type 2 (AT2) receptor blocker on bone mass. Immunohistochemistry revealed that AT2 receptor protein was expressed in both osteoblasts and osteoclasts. In addition, renin and angiotensin II-converting enzyme were expressed in bone cells in vivo. Treatment with AT2 receptor blocker significantly enhanced the levels of bone mass, and this effect was based on the enhancement of osteoblastic activity as well as the suppression of osteoclastic activity in vivo. These results indicate that RAS components are present in adult bone and that blockade of AT2 receptor results in alteration in bone mass.

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

BackgroundConditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.ResultsHere we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.ConclusionsEasi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Sympathetic control of bone mass regulated by osteopontin

The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the β2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the β2-adrenergic receptor/cAMP signaling system.

PlexinD1 deficiency induces defects in axial skeletal morphogenesis.

Axial patterning in embryonic skeletogenesis associates with coordinated programming of somitogenesis and angiogenesis. As seen in endochondral bone formation, skeletogenesis is closely related to angiogenesis during development. PlexinD1 is a member of plexin family, is expressed in central nervous system and endothelium, and plays a role in blood vessel patterning and endothelium positioning during embryonic development. Here, we examined the effects of PlexinD1 deficiency on skeletogenesis. Three‐dimensional micro CT examination revealed that PlexinD1 deficiency resulted in axial skeletal patterning defects including malformation in vertebral body and rib bone shape. Histological examination of the vertebral bodies and long bones showed that PlexinD1 deficiency altered the development of cartilage. PlexinD1 deficiency did not affect the levels of von Willebrand factor staining in relatively large vessels not attached but close to the vertebral body of mice. However, PlexinD1 deficiency reduced the von Willebrand factor (vWf) staining in most of the microvasculatures attached to the vertebral bone. PlexinD1 was expressed in osteoblastic cells and bone tissues of newborn and adult mice. As most of the homozygous knockout mice did not survive, we examined the role of PlexinD1 in bone formation in heterozygous adult mice subjected to bone marrow ablation. However, PlexinD1 heterozygous knockout did not reveal defects in new bone formation. In conclusion, PlexinD1 is involved in the patterning of axial skeletogenesis. J. Cell. Biochem. 101: 1329–1337, 2007.

JunD Suppresses Bone Formation and Contributes to Low Bone Mass Induced by Estrogen Depletion

JunD is an activator protein‐1 (AP‐1) component though its function in skeletal system is still not fully understood. To elucidate the role of JunD in the regulation of bone metabolism, we analyzed JunD‐deficient mice. JunD deficiency significantly increased bone mass and trabecular number. This bone mass enhancement was due to JunD deficiency‐induced increase in bone formation activities in vivo. Such augmentation of bone formation was associated with simultaneous increase in bone resorption while the former was dominant over the latter as accumulation of bone mass occurred in JunD‐deficient mice. In a pathological condition relevant to postmenopausal osteoporosis, ovariectomy reduced bone mass in wild type (WT) mice as known before. Interestingly, JunD deficiency suppressed ovariectomy‐induced increase in bone resorption and kept high bone mass. In addition, JunD deficiency also enhanced new bone formation after bone marrow ablation. Examination of molecular bases for these observations revealed that JunD deficiency enhanced expression levels of c‐jun, fra‐1, and fra‐2 in bone in conjunction with elevated expression levels of runx2, type I collagen, and osteocalcin. Thus, JunD is involved in estrogen depletion‐induced osteopenia via its action to suppress bone formation and to enhance bone resorption. J. Cell. Biochem. 103: 1037–1045, 2008.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

BackgroundAlthough CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining.ResultsHere, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes.ConclusionsOur results provide a technical platform for high-throughput knock-in.

ANA Deficiency Enhances Bone Morphogenetic Protein-induced Ectopic Bone Formation via Transcriptional Events

Ectopic bone formation after joint replacement or brain injury in humans is a serious complication that causes immobility of joints and severe pain. However, mechanisms underlying such ectopic bone formation are not fully understood. Bone morphogenetic protein (BMPs) are defined as inducers of ectopic bone formation, and they are regulated by several types of inhibitors. ANA is an antiproliferative molecule that belongs to Tob/BTG family, but its activity in bone metabolism has not been known. Here, we examined the role of ANA on ectopic bone formation activity of BMP. In ANA-deficient and wild-type mice, BMP2 was implanted to induce ectopic bone formation in muscle. ANA deficiency increased mass of newly formed bone in vivo compared with wild-type based on 3D-μCT analyses. ANA mRNA was expressed in bone in vivo as well as in osteoblastic cells in vitro. Such ANA mRNA levels were increased by BMP2 treatment in MC3T3-E1 osteoblastic cells. Overexpression of ANA suppressed BMP-induced expression of luciferase reporter gene linked to BMP response elements in these cells. Conversely, ANA mRNA knockdown by small interference RNA enhanced the BMP-dependent BMP response element reporter expression. It also enhanced BMP-induced osteoblastic differentiation in muscle-derived C2C12 cells. Immunoprecipitation assay indicated that ANA interacts with Smad8. Thus, ANA is a suppressor of ectopic bone formation induced by BMP, and this inhibitory ANA activity is a part of the negative feedback regulation of BMP function.

Type VI collagen deficiency induces osteopenia with distortion of osteoblastic cell morphology

Bone consists of type I collagen as a major protein with minor various matrix proteins. Type VI collagen is one of bone matrix proteins but its function is not known. We therefore examined the effects of type VI collagen deficiency on bone. 3D-μCT analysis revealed that type VI collagen deficiency reduced cancellous bone mass. Cortical bone mass was not affected. Type VI collagen deficiency distorted the shape of osteoblasts both in the cancellous bone and in the cambium layer of periosteal region. Furthermore, type VI collagen deficiency disorganized collagen arrangement. These data indicate that type VI collagen contributes to maintain bone mass.

Collagens VI and XII form complexes mediating osteoblast interactions during osteogenesis

Bone formation is precisely regulated by cell-cell communication in osteoblasts. We have previously demonstrated that genetic deletion of Col6a1 or Col12a1 impairs osteoblast connections and/or communication in mice, resulting in bone mass reduction and bone fragility. Mutations of the genes encoding collagen VI cause Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), which have overlapping phenotypes involving connective tissue and muscle. Recent studies have identified COL12A1 gene mutations in patients with UCMD- and BM-like disorders harboring no COL6 mutations, indicating the shared functions of these collagens in connective tissue homeostasis. The purpose of this investigation has been to test the hypothesis that collagens VI and XII have coordinate regulatory role(s) during bone formation. We analyzed the localization of collagens VI and XII relative to primary osteoblasts during osteogenesis. Immunofluorescence analysis demonstrated that collagens VI and XII colocalized in matrix bridges between adjacent cells during periods when osteoblasts were establishing cell-cell connections. Quantification of cells harboring collagen bridges demonstrated that matrix bridges were composed of collagens VI and XII but not collagen I. Interestingly, matrix bridge formation was impaired in osteoblasts deficient in either Col6a1 or Col12a1, suggesting that both collagens were indispensable for matrix bridge formation. These data demonstrate, for the first time, a functional relationship between collagens VI and XII during osteogenesis and indicate that a complex containing collagens VI and XII is essential for the formation of a communicating cellular network during bone formation.