The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

Epigenetic modifier G9a is involved in regulation of mouse tongue development.

OBJECTIVES: The tongue comprises multiple tissues of different embryonic origins, including pharyngeal arch, somite, and cranial neural crest (CNC). However, its developmental regulatory mechanisms, especially those involving epigenetic modifiers, remain poorly understood. This study examined the roles of the epigenetic modifier G9a in murine tongue development. METHODS: We deleted G9a using Sox 9 (SRY-related HMG-box gene 9)-Cre recombinase, which acts in tongue progenitor cells, including CNC-derived cells, to generate G9a conditional knockout (cKO) mice. Histochemical and immunohistochemical analyses were conducted on sections prepared from tongue tissues of control and cKO mice. RESULTS: Cre-dependent LacZ reporter mice, generated by crossing Rosa-LacZ mice with sox9-Cre mice, revealed Cre recombinase activity in the mucosal epithelium and tongue connective tissue of the embryonic tongue. Tongue volume was significantly reduced on embryonic day 17.5 (E17.5) and postnatal day 0 (P0) in cKO mice. Histological sections showed that the lingual mucosal epithelium was thinner in cKO mice. Reduced G9a levels were accompanied by decreased levels of a G9a substrate, dimethylated lysine 9 in histone H3, in the embryonic tongue. BrdU injection at E16.5 revealed reduced numbers of BrdU-positive cells in the mucosal epithelium and underlying connective tissue at E17.5 in cKO mice, indicating suppression of cell proliferation in both tissues. Investigation of keratin 5 and 8 protein localization showed significantly suppressed expression in the lingual mucosal epithelium in cKO mice. CONCLUSIONS: G9a is required for proper proliferation and differentiation of sox9-expressing tongue progenitor cells and is thereby involved in tongue development.

Tooth transplantation and replantation: Biological insights towards therapeutic improvements.

Transplantation and replantation of teeth are effective therapeutic approaches for tooth repositioning and avulsion, respectively. Transplantation involves transplanting an extracted tooth from the original site into another site, regenerating tissue including the periodontal ligament (PDL) and alveolar bone, around the transplanted tooth. Replantation places the avulsed tooth back to its original site, regenerating functional periodontal tissue. In clinical settings, transplantation and replantation result in favorable outcomes with regenerated PDL tissue in many cases. However, they often result in poor outcomes with two major complications: tooth ankylosis and root resorption. In tooth ankylosis, the root surface and alveolar bone are fused, reducing the PDL tissue between them. The root is subjected to remodeling processes and is partially replaced by bone. In severe cases, the resorbed root is completely replaced by bone tissue, which is called as "replacement resorption." Resorption is sometimes accompanied by infection-mediated inflammation. The molecular mechanisms of ankylosis and root resorption remain unclear, although some signaling mechanisms have been proposed. In this mini-review, we summarized the biological basis of repair mechanisms of tissues in transplantation and replantation and the pathogenesis of their healing failure. We also discussed possible therapeutic interventions to improve treatment success rates.

Platelet-derived growth factor-BB regenerates functional periodontal ligament in the tooth replantation.

Tooth ankylosis is a pathological condition of periodontal ligament (PDL) restoration after tooth replantation. Platelet-derived growth factor-BB (PDGF-BB) has been proposed as a promising factor for preventing tooth ankylosis. Using rat tooth replantation model, we investigated whether PDGF-BB accelerates the repair of PDL after tooth replantation without ankylosis, and its molecular mechanisms. In PDGF-BB pretreated replanted teeth (PDGF-BB group), ankylosis was markedly reduced and functionally organized PDL collagen fibers were restored; the mechanical strength of the healing PDL was restored to an average of 76% of that in non-replanted normal teeth at 21 days. The numbers of PDGF-Rß- and BrdU-positive cells in the periodontal tissues of the PDGF-BB group were greater than those of atelocollagen pretreated replanted teeth (AC group). Moreover, in the PDGF-BB group, the periodontal tissues had fewer osteocalcin-positive cells and decreased number of nuclear ß-catenin-positive cells compared to those in the AC group. In vitro analyses showed that PDGF-BB increased the proliferation and migration of human periodontal fibroblasts. PDGF-BB downregulated mRNA expressions of RUNX2 and ALP, and inhibited upregulatory effects of Wnt3a on ß-catenin, AXIN2, RUNX2, COL1A1, and ALP mRNA expressions. These findings indicate that in tooth replantation, topical PDGF-BB treatment enhances cell proliferation and migration, and inhibits canonical Wnt signaling activation in bone-tooth ankylosis, leading to occlusal loading of the PDL tissues and subsequent functional restoration of the healing PDL. This suggests a possible clinical application of PDGF-BB to reduce ankylosis after tooth replantation and promote proper regeneration of PDL.

G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development.

The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9aflox/flox (fl/fl) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9afl/fl mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.

Annexin A5 Involvement in Bone Overgrowth at the Enthesis.

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5-/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.

Essential roles of G9a in cell proliferation and differentiation during tooth development.

Teeth develop through interactions between epithelial and mesenchymal tissues mediated by a signaling network comprised of growth factors and transcription factors. However, little is known about how epigenetic modifiers affect signaling pathways and thereby regulate tooth formation. We previously reported that the histone 3 lysine 9 (H3K9) methyltransferase (MTase) G9a is specifically enriched in the tooth mesenchyme during mouse development. In this study, we investigated the functions of G9a in tooth development using G9a conditional knockout (KO) mice. We used Sox9-Cre mice to delete G9a in the tooth mesenchyme because Sox9 is highly expressed in the mesenchyme derived from the cranial neural crest. Immunohistochemical analyses revealed that G9a expression was significantly decreased in the mesenchyme of Sox9-Cre;G9afl/fl (G9a cKO) mice compared with that in Sox9-Cre;G9a fl/+(control) mice. Protein levels of the G9a substrate H3K9me2 were also decreased in the tooth mesenchyme. G9a cKO mice showed smaller tooth germ after embryonic day (E) 16.5 and E17.5, but not at E15.5. The developing cusp tips, which were visible in control mice, were absent in G9a cKO mice at E17.5. At 3 weeks after birth, small first molars with smaller cusps and unseparated roots were formed. Organ culture of tooth germs derived from E15.5 cKO mouse embryos showed impaired tooth development, suggesting that tooth development per se is affected independently of skull development. BrdU labeling experiments revealed that the proliferation rates were decreased in the mesenchyme in G9a cKO mice at E17.5. In addition, the proliferation rates in the tooth inner enamel epithelium were also decreased. In situ hybridization revealed altered localization of genes associated with tooth development. In cKO mice, intensively localized expression of mRNAs encoding bone morphogenic protein (Bmp2 and Bmp4) was observed in the tooth mesenchyme at E17.5, similar to the expression patterns observed in control mice at E15.5. Localization of Shh and related signaling components, including Gli1, Ptch1, and Ptch2, in the tooth mesenchyme of cKO mice was generally similar to that at earlier stages in control mice. In addition, expression of Fgf3 and Fgf10 in the mesenchyme was decreased in G9a cKO mice at P0. Expression levels of Fgf9 and p21, both of which were expressed in the secondary enamel-knot, were also decreased. Thus, the expression of genes associated with tooth development was delayed in cKO mice. Our results suggest that H3K9MTase G9a regulates cell proliferation and timing of differentiation and that G9a expression in the tooth mesenchyme is required for proper tooth development.

Bardet-Biedl syndrome 3 regulates the development of cranial base midline structures.

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder and is classified as one of the ciliopathy. The patients manifest a characteristic craniofacial dysmorphology but the effects of Bbs3 deficiency in the developmental process during the craniofacial pathogenesis are still incompletely understood. Here, we analyzed a cranial development of a BBS model Bbs3-/- mouse. It was previously reported that these mutant mice exhibit a dome-shape cranium. We show that Bbs3-/- mouse embryos present mid-facial hypoplasia and solitary central upper incisor. Morphologically, these mutant mice show synchondrosis of the cranial base midline due to the failure to fuse in association with loss of intrasphenoidal synchondrosis. The cranial base was laterally expanded and longitudinally shortened. In the developing cartilaginous primordium of cranial base, cells present in the midline were less in Bbs3-/- embryos. Expression of BBS3 was observed specifically in a cell population lying between condensed ectomesenchyme in the midline and the ventral midbrain at this stage. Finally, siRNA-based knockdown of Bbs3 in ATDC5 cells impaired migration in culture. Our data suggest that BBS3 is required for the development of cranial base via regulation of cell migration toward the midline where they promote the condensation of ectomesenchyme and form the future cartilaginous templates of cranial base.

H3K9MTase G9a is essential for the differentiation and growth of tenocytes in vitro.

Cell differentiation is controlled by specific transcription factors. The functions and expression levels of these transcription factors are regulated by epigenetic modifications, such as histone modifications and cytosine methylation of the genome. In tendon tissue, tendon-specific transcription factors have been shown to play functional roles in the regulation of tenocyte differentiation. However, the effects of epigenetic modifications on gene expression and differentiation in tenocytes are unclear. In this study, we investigated the epigenetic regulation of tenocyte differentiation, focusing on the enzymes mediating histone 3 lysine 9 (H3K9) methylation. In primary mouse tenocytes, six H3K9 methyltransferase (H3K9MTase) genes, i.e., G9a, G9a-like protein (GLP), PR domain zinc finger protein 2 (PRDM2), SUV39H1, SUV39H2, and SETDB1/ESET were all expressed, with increased mRNA levels observed during tenocyte differentiation. In mouse embryos, G9a and Prdm2 mRNAs were expressed in tenocyte precursor cells, which were overlapped with or were adjacent to cells expressing a tenocyte-specific marker, tenomodulin. Using tenocytes isolated from G9a-flox/flox mice, we deleted G9a by infecting the cells with Cre-expressing adenoviruses. Proliferation of G9a-null tenocytes was significantly decreased compared with that of control cells infected with GFP-expressing adenoviruses. Moreover, the expression levels of tendon transcription factors gene, i.e., Scleraxis (Scx), Mohawk (Mkx), Egr1, Six1, and Six2 were all suppressed in G9a-null tenocytes. The tendon-related genes Col1a1, tenomodulin, and periostin were also downregulated. Consistent with this, Western blot analysis showed that tenomodulin protein expression was significantly suppressed by G9a deletion. These results suggested that expression of the H3K9MTase G9a was essential for the differentiation and growth of tenocytes and that H3K9MTases may play important roles in tendinogenesis.

Coordinated expression of H3K9 histone methyltransferases during tooth development in mice.

Tissue-specific gene expression is subjected to epigenetic and genetic regulation. Posttranslational modifications of histone tails alter the accessibility of nuclear proteins to DNA, thus affecting the activity of the regulatory complex of nuclear proteins. Methylation at histone 3 lysine 9 (H3K9) is a crucial modification that affects gene expression and cell differentiation. H3K9 is known to have 0-3 methylation states, and these four methylated states are determined by the expression of sets of histone methyltransferases. During development, teeth are formed through mutual interactions between the mesenchyme and epithelium via a process that is subjected to the epigenetic regulation. In this study, we examined the expression of all H3K9 methyltransferases (H3K9MTases) during mouse tooth development. We found that four H3K9MTases-G9a, Glp, Prdm2, and Suv39h1-were highly expressed in the tooth germ, with expression peaks at around embryonic days 16.5 and 17.5 in mice. Immunohistochemical and in situ hybridization analyses revealed that all four H3K9MTases were enriched in the mesenchyme more than in the epithelium. Substrates of H3K9MTases, H3K9me1, H3K9me2, and H3K9me3 were also enriched in the mesenchyme. Taken together, these data suggested that coordinated expression of four H3K9MTases in the dental mesenchyme might play important roles in tooth development.

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method.

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell-cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.

Alendronate promotes bone formation by inhibiting protein prenylation in osteoblasts in rat tooth replantation model.

Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.

Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.

The advantages of using induced pluripotent stem cells (iPSCs) instead of embryonic stem (ES) cells in regenerative medicine centre around circumventing concerns about the ethics of using ES cells and the likelihood of immune rejection of ES-cell-derived tissues. However, partial reprogramming and genetic instabilities in iPSCs could elicit immune responses in transplant recipients even when iPSC-derived differentiated cells are transplanted. iPSCs are first differentiated into specific types of cells in vitro for subsequent transplantation. Although model transplantation experiments have been conducted using various iPSC-derived differentiated tissues and immune rejections have not been observed, careful investigation of the immunogenicity of iPSC-derived tissue is becoming increasingly critical, especially as this has not been the focus of most studies done so far. A recent study reported immunogenicity of iPSC- but not ES-cell-derived teratomas and implicated several causative genes. Nevertheless, some controversy has arisen regarding these findings. Here we examine the immunogenicity of differentiated skin and bone marrow tissues derived from mouse iPSCs. To ensure optimal comparison of iPSCs and ES cells, we established ten integration-free iPSC and seven ES-cell lines using an inbred mouse strain, C57BL/6. We observed no differences in the rate of success of transplantation when skin and bone marrow cells derived from iPSCs were compared with ES-cell-derived tissues. Moreover, we observed limited or no immune responses, including T-cell infiltration, for tissues derived from either iPSCs or ES cells, and no increase in the expression of the immunogenicity-causing Zg16 and Hormad1 genes in regressing skin and teratoma tissues. Our findings suggest limited immunogenicity of transplanted cells differentiated from iPSCs and ES cells.

Predominant expression of H3K9 methyltransferases in prehypertrophic and hypertrophic chondrocytes during mouse growth plate cartilage development.

Histone lysine methylation (HKM) is an epigenetic change that establishes cell-specific gene expression and determines cell fates. In this study, we investigated the expression patterns of histone H3 lysine 9 methyltransferases (H3K9MTases) G9a (euchromatic histone lysine N-methyltransferase 2, Ehmt2), GLP (euchromatic histone lysine N-methyltransferase 1, Ehmt1), SETDB1 (SET domain, bifurcated 1), PRDM2 (PR domain containing 2), SUV39H1 (suppressor of variegation 3-9 homolog 1), and SUV39H2, as well as the distribution of 3 types of HKM at histone H3 lysine 9: mono- (H3K9me1), di- (H3K9me2), or tri-methylation (H3K9me3), during mouse growth plate development. In the forelimb cartilage primordial at embryonic day 12.5 (E12.5), none of the H3K9MTases were detected and H3K9me1, H3K9me2, and H3K9me3 were scarcely detected. At E14.5, the H3K9MTases were expressed at low levels in proliferating chondrocytes and at high levels in prehypertrophic and hypertrophic chondrocytes. Among the H3K9 methylations, H3K9me1 and H3K9me3 were markedly noted in these chondrocytes. At E16.5, G9, GLP, SETDB1, PRDM2, SUV39H1, and SUV39H2, as well as H3K9me1, H3K9me2, and H3K9me3, were detected in prehypertrophic and hypertrophic chondrocytes in the growth plate. Western blotting and real-time quantitative polymerase chain reaction analysis revealed the distributions of G9 and GLP proteins and the expression of all the H3K9MTase mRNAs in prehypertrophic and hypertrophic chondrocytes. These data suggest that H3K9 methyltransferases are predominantly expressed in prehypertrophic and hypertrophic chondrocytes, and that they could be involved in the regulation of gene expression and progression of chondrocyte differentiation by affecting the methylation state of histone H3 lysine 9 in the mouse growth plate.

Age-related effects of fasting on ketone body production during lipolysis in rats.

OBJECTIVE: The age-related effects of fasting on lipolysis, the production of ketone bodies, and plasma insulin levels were studied in male 3-, 8-, and 32-week-old Sprague-Dawley rats. METHODS: The rats were divided into fasting and control groups. The 3-, 8- and 32-week-old rats tolerated fasting for 2, 5, and 12 days, respectively. RESULTS: Fasting markedly reduced the weights of perirenal and periepididymal white adipose tissues in rats in the three age groups. The mean rates of reduction in both these adipose tissue weights during fasting periods were higher in the order of 3 > 8 > 32-week-old rats. Fasting transiently increased plasma free fatty acid (FFA), total ketone body, ß-hydroxybutyrate, and acetoacetate concentrations in the rats in the three age groups. However, plasma FFA, total ketone body, ß-hydroxybutyrate, and acetoacetate concentrations in the 3-week-old rats reached maximal peak within 2 days after the onset of fasting, although these concentrations in the 8- and 32-week-old rats took more than 2 days to reach the maximal peak. By contrast, the augmentation of plasma FFA, total ketone body, ß-hydroxybutyrate, and acetoacetate concentrations in the rats in the three age groups had declined at the end of each experimental period. Thus, the capacity for fat mobilization was associated with tolerance to fasting. Plasma insulin concentrations in the rats in the three age groups were dramatically reduced during fasting periods, although basal levels of insulin were higher in the order of 32 > 8 > 3 week-old rats. CONCLUSION: These results suggest that differences in fat metabolism patterns among rats in the three age groups during prolonged fasting were partly reflected the metabolic turnover rates, plasma insulin levels, and amounts of fat storage.

Dietary zinc-deficiency and its recovery responses in the thermogenesis of rats.

The aim of this study was to elucidate whether dietary zinc-deficiency and its recovery play a role in controlling autonomic thermoregulation. We investigated the effects of dietary zinc-deficiency and its recovery on autonomic thermoregulation by measuring the rectal temperature, an index of deep body temperature. The weaned male Sprague Dawley rats were randomly divided into the dietary zinc-deficient diet (0.6 mg zinc/kg diet) group and the control diet (35.2 mg zinc/kg diet) group, and were fed for 4 weeks. In the recovery period, the rats of two groups were fed with the control diet for 3 weeks. The rectal temperature was significantly decreased throughout the period of zinc-deficiency and the hypothermic responses during the experimental period were recovered to the control group levels at least within 1 week in the recovery process from dietary zinc-deficiency. These results suggest that the deep temperature in rats was clearly associated with the dietary zinc intake levels.

Single administration effects of ethanol on the distribution of white blood cells in rats.

Acute single administration effects of ethanol on the distribution of total white blood cells (WBCs), neutrophil, basophil, eosinophil, monocyte and lymphocytes were studied in rats. Acute single administration effects of ethanol on the number of red blood cells (RBCs), hemoglobin concentration and hematocrit were also examined. Male 8-week-old Sprague Dawley rats were randomly divided into the ethanol-administered (ETA) group and the control (CON) group. Two parts of an experiment, 1) 1st experiment : (ethanol dose : 1.0 g/kg body weight), and 2) 2nd experiment : (ethanol dose : 2.0 g/kg body weight) were carried out in rats. The rats were starved to 19:00, and deprived of diet for 12 hr and water for 1 hr before the single administration of ethanol. 1.0 or 2.0 g/kg body weight of ethanol (in 20% (w/w) ethanol) was orally administered to ETA group rats via a stainless stomach tube. In the CON group rats, 0.9% NaCl solution was orally given with the solution volume being equal, in the same manner. Single administration of 1.0 or 2.0 g/kg body weight of ethanol did not change the number of RBCs, hemoglobin concentration and hematocrit. Single administration of 1.0 g/kg body weight of ethanol did not also change the number of WBCs. However, administration of 2.0 g/kg body weight of ethanol increased significantly the number of neutrophil, basophil, monocyte and total WBCs without changing the number of eosinophil and lymphocytes. These results suggest that single administration of 2.0 g/kg body weight of ethanol to rats increased markedly the number of the natural immunity cells without changing the number of acquired cells.

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation.

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLK in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.

Protein related to DAN and cerberus (PRDC) inhibits osteoblastic differentiation and its suppression promotes osteogenesis in vitro.

Protein related to DAN and cerberus (PRDC) is a secreted protein characterized by a cysteine knot structure, which binds bone morphogenetic proteins (BMPs) and thereby inhibits their binding to BMP receptors. As an extracellular BMP antagonist, PRDC may play critical roles in osteogenesis; however, its expression and function in osteoblastic differentiation have not been determined. Here, we investigated whether PRDC is expressed in osteoblasts and whether it regulates osteogenesis in vitro. PRDC mRNA was found to be expressed in the pre-osteoblasts of embryonic day 18.5 (E18.5) mouse calvariae. PRDC mRNA expression was elevated by treatment with BMP-2 in osteoblastic cells isolated from E18.5 calvariae (pOB cells). Forced expression of PRDC using adenovirus did not affect cell numbers, whereas it suppressed exogenous BMP activity and endogenous levels of phosphorylated Smad1/5/8 protein. Furthermore, PRDC inhibited the expression of bone marker genes and bone-like mineralized matrix deposition in pOB cells. In contrast, the reduction of PRDC expression by siRNA elevated alkaline phosphatase activity, increased endogenous levels of phosphorylated Smad1/5/8 protein, and promoted bone-like mineralized matrix deposition in pOB cells. These results suggest that PRDC expression in osteoblasts suppresses differentiation and that reduction of PRDC expression promotes osteogenesis in vitro. PRDC is accordingly identified as a potential novel therapeutic target for the regulation of bone formation.