RGS12 is essential for RANKL-evoked signaling for terminal differentiation of osteoclasts in vitro.

How RANKL evokes [Ca(2+)](i) oscillations and leads to osteoclast differentiation is unclear. We identified a new signaling protein, RGS12, and found that RGS12 is essential for [Ca(2+)](i) oscillations and osteoclast differentiation induced by RANKL. RGS12 may play a critical role in the RANKL-evoked PLCgamma-calcium channels-[Ca(2+)](i) oscillation-NFAT2 pathway. INTRODUCTION: RANKL-induced [Ca(2+)](i) oscillations play a switch-on role in NFAT2 expression and osteoclast differentiation. However, RANKL evokes [Ca(2+)](i) oscillations and leads to osteoclast differentiation by an unknown mechanism. In this study, we identified a new RANKL-induced signaling protein, regulator of G signaling protein 12 (RGS12), and investigated its effect on osteoclast differentiation in vitro. MATERIALS AND METHODS: We used a genome-wide screening approach to identify genes that are specifically or prominently expressed in osteoclasts. To study the role of the RGS12 in osteoclast differentiation, we used vector and lentivirus-based RNAi gene silencing technology to silence the RGS12 gene in the monocyte progenitor cell lines and primary bone marrow-derived monocytes (BMMs). The interaction between RGS12 and N-type calcium channels was elucidated using co-immunoprecipitation and immunoblotting. RESULTS: We found that RGS12 was prominently expressed in osteoclast-like cells (OLCs) induced by RANKL. This result was further confirmed at both the mRNA and protein level in human osteoclasts and mouse OLCs. Silence of RGS12 expression using vector and lentivirus based RNA interference (RNAi) impaired phosphorylation of phospholipase C (PLC)gamma and blocked [Ca(2+)](i) oscillations, NFAT2 expression, and osteoclast differentiation in RANKL-induced RAW264.7 cells and BMMs. We further found that N-type calcium channels were expressed in OLCs after RANKL stimulation and that RGS12 directly interacted with the N-type calcium channels. CONCLUSIONS: These results reveal that RGS12 is essential for the terminal differentiation of osteoclasts induced by RANKL. It is possible that RGS12 regulates osteoclast differentiation through a PLC gamma-calcium channel-[Ca(2+)](i) oscillation-NFAT2 pathway.     

The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts

Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance because actin cytoskeleton is essential to generate the sealing zone, an osteoclast‐specific structure ultimately mediating bone resorption. Here we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We discovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small‐molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5^–/– mice and found that they have increased trabecular bone mass with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop antiosteoporotic treatments. © 2011 American Society for Bone and Mineral Research.     

Tartrate-resistant acid phosphatase is not an exclusive marker for mouse osteoclasts in cell culture

The method of Barka and Anderson was used for the demonstration of tartrate-resistant acid phosphatase (TRAcP) in cultures of bone marrow, spleen, lung, and peritoneal cells of the mouse. The staining was performed either in the usual way by adding both substrate (naphthol-AS-BI-phosphate) and coupler (hexazonium pararosanilin) together (the simultaneous-coupling technique) or by adding first the substrate and then the coupler (the post-coupling technique). We measured TRAcP-activity fluorometrically after extraction of the product naphthol-AS-BI, using the same staining solution as in cytochemical method, but without the coupler. In bone marrow, spleen, lung, and peritoneal cell cultures a biochemically measurable TRAcP-activity was detected. Post-coupling generally gave a higher level of staining and larger numbers of TRAcP-positive cells than simultaneous-coupling. In bone marrow cultures macrophages, identifiable by their ability to phagocytose microspheres, became TRAcP-positive during culture. In lung cell cultures cells capable of phagocytosis of bacteria were shown to be TRAcP-positive. Peritoneal macrophages remained TRAcP-negative in the simultaneous-coupling technique. Using the post-coupling technique a small number stained TRAcP-positive. In spleen cell cultures TRAcP-positive cells containing hemosiderin were visible. In cultures of all four cell types, F4/80 positive cells staining also for TRAcP were present. F4/80 is a well known marker for macrophages, whereas osteoclasts are negative. In conclusion, mouse macrophages originating from various tissues can become TRAcP-positive in vitro. TRAcP activity alone is not a reliable marker for osteoclasts in bone marrow cultures.     

The origin of osteoclasts: An immunohistochemical study on macrophages and osteoclasts in embryonic rat bone

Summary The origin of osteoclasts was studied in embryonic rat bone primordia using a set of monoclonal antibodies (ED1, ED2, and ED3) that exclusively recognize monocytes and macrophages. ED1 recognizes monocytes and macrophages. Mononuclear phagocytes which were ED1 positive were found in the perichondrium/periosteum of developing bone. These cells started to infiltrate the primordia when the cartilage became hypertrophic. During bone formation, multinucleated ED1-positive cells with the morphological characteristics of osteoclasts were found in the developing bone marrow cavity and against the bone collar. The present findings support the notion that osteoclasts arise by fusion of mononuclear phagocytes derived from blood monocytes.     

End of embryonic somite period in the rat and a comparison with the corresponding embryonic period in humans]

The end of the embryonic somite period--stage 12 according of the Carnegie Institution--was identified in the rat. The series was composed of 23 embryos taken out from uterus on day 11/4H, day 11/5H and day 11/10H post fertilization. The specimens were submitted to external macroscopic examinations and to histological sections. The rat development of this stage was characterized by the closure of the caudal neuropore, the evidence of 3 pharyngeal archs, the closure of the otic vesicle, the presence of the fore limb buds, the beginning of the cardiac septa. This stage, being similar to this of human embryos, the authors concluded that rat could be an excellent comparative model for experimental Teratology.     

Nitrofen inhibition of pulmonary growth and development occurs in the early embryonic mouse

Background/Purpose: It was believed previously that pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) was a consequence of the herniation of abdominal viscera into the chest. Using the murine nitrofen-induced model of CDH, the authors evaluated lung growth and development before diaphragm closure or herniation. Methods: The authors examined nitrofen-exposed early embryonic lungs on embryonic day 12 (E12). Branching morphogenesis was quantified before and after 4 days in culture in serumless chemically defined media and compared with age-matched control lungs. The mRNA expression of proliferative and developmental markers in cultured lungs was then determined. Results: Nitrofen-exposed lungs had 30% fewer total terminal branches than age-matched controls (9.3 [plusmn] 1.9 nitrofen v 13.7 [plusmn] 2.6 control; P [lt ] .001). Hypoplasia also was more profound in the left than the right lung. These effects persisted after culturing the lungs for 4 days in serumless chemically-defined media (31.7 [plusmn] 6.8 nitrofen v 42.9 [plusmn] 8.4 control, P [lt ] .001). Furthermore, the mRNA expression of proliferative and developmental markers was decreased in nitrofen-exposed E12 lungs cultured for 4 days (as a percentage of age-matched controls): cyclin A (69.28%; P = .04), Nkx2.1 (44.4%, 0.04), SP-A (24.1%; P = .008), SP-B (23.4%; P = .05), SP-C (20%; P = .06), and CC-10 (13.8%; P = .04). Conclusion: Nitrofen induces primary pulmonary hypoplasia and immaturity in the early embryonic mouse, and this effect persists in culture. J Pediatr Surg 37:1263-1268.     

Spontaneous tooth eruption after alveolar cleft osteoplasty using tissue-engineered bone: a case report.

To bridge the cleft in the alveolar bone and to allow for physiologic eruption of the canine teeth, alveolar bone grafting is often necessary in patients with cleft lips and palates. Instead of autogenous bone, biomaterial seeded with autogenous osteogenic cells has found some clinical application. However, so far no real functional proof has been available to demonstrate that this technique also allows further physiologic features such as tooth eruption to occur. This report describes the results of grafting tissue-engineered bone into the alveolar cleft of a 10-year-old boy. Immediate postoperative healing was uneventful. Eight months after grafting, erupting teeth had moved into the newly formed bone. Eighteen months postoperatively at the site where the tissue-engineered graft had been inserted, the canine had erupted spontaneously in its proper place. The data suggest that tissue-engineered bone can lead to the ossification of the alveolar cleft and allow for physiologic spontaneous tooth eruption.     

Hepatic glucagon action is essential for exercise-induced reversal of mouse fatty liver

OBJECTIVE

Exercise is an effective intervention to treat fatty liver. However, the mechanism(s) that underlie exercise-induced reductions in fatty liver are unclear. Here we tested the hypothesis that exercise requires hepatic glucagon action to reduce fatty liver.

RESEARCH DESIGN AND METHODS

C57BL/6 mice were fed high-fat diet (HFD) and assessed using magnetic resonance, biochemical, and histological techniques to establish a timeline for fatty liver development over 20 weeks. Glucagon receptor null (gcgr^−/−) and wild-type (gcgr^+/+) littermate mice were subsequently fed HFD to provoke moderate fatty liver and then performed either 10 or 6 weeks of running wheel or treadmill exercise, respectively.

RESULTS

Exercise reverses progression of HFD-induced fatty liver in gcgr^+/+ mice. Remarkably, such changes are absent in gcgr^−/− mice, thus confirming the hypothesis that exercise-stimulated hepatic glucagon receptor activation is critical to reduce HFD-induced fatty liver.

CONCLUSIONS

These findings suggest that therapies that use antagonism of hepatic glucagon action to reduce blood glucose may interfere with the ability of exercise and perhaps other interventions to positively affect fatty liver.Nonalcoholic fatty liver (fatty liver) is a disease characterized by lipid infiltration in hepatocytes. Fatty liver is important to identify and treat because it may progress to more severe dysfunction and is linked to increased mortality (1). Fatty liver is also independently associated with obesity, type 2 diabetes, and insulin resistance and is an emerging component of metabolic syndrome (1). Studies in humans (2–5) and rodents (6–12) show that exercise positively affects fatty liver and/or measures of hepatic function in a manner partially independent of weight loss. It is unclear, however, how exercise causes such improvements. One mechanism that may underlie exercise-induced reductions in fatty liver is repeat bouts of hepatic glucagon action. Exercise stimulates a rise in hepatic glucagon action that spurs pathways that acutely fuel glucose production (13–18). Exercise-induced increases in glucagon action also provoke changes in hepatic gene expression consistent with chronically elevated fat oxidation (19).The current focus was to test whether exercise-induced reductions in high-fat diet (HFD)-induced fatty liver require hepatic glucagon action. Male C57BL/6 (BL6) mice were first fed HFD to establish a timeline for development of fatty liver. A magnetic resonance (MR) technique was also developed to perform noninvasive measures of liver fat. Mice with intact glucagon receptors (gcgr^+/+) and null littermates (gcgr^−/−) were then fed HFD to induce moderate fatty liver and subsequently housed with running wheels to promote voluntary physical activity or performed treadmill exercise to standardize the dose. Collectively, these data establish a timeline for HFD-induced fatty liver in BL6 mice and provide mechanistic detail to understand how exercise and hepatic glucagon action interact to reduce fatty liver.     

Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts

The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.     

RhBMP-7 improves survival and eruption in a growing tooth avulsion trauma model

Long-term loss of avulsed and replanted teeth is a frequent clinical problem. Bone morphogenetic protein-7 (BMP 7) induces cementogenesis in periodontitis-associated periodontal ligament (PDL) defects. This study's aim was to assess the utility of rhBMP 7 in a tooth avulsion trauma model in growing individuals. Immature primary incisors of 12 minipigs were extracted. PDL and cementum were removed either partially (group 1: 4 mm2 [n=28 teeth]; group 2: 16 mm2 [n=26 teeth]) or totally (group 3 [n=26 teeth]). 500 microg rhBMP 7/g collagen matrix was applied to the teeth from one side while the corresponding teeth on the contralateral side served as controls (split mouth model). After an experimental period of 4 months, microradiography, fluorescence and light microscopy of nondecalcified sections were performed. All teeth of group 1 survived and all teeth of group 3 were lost, whether rhBMP-7 was applied or not. In group 2, nine out of ten teeth survived when rhBMP-7 was applied and four out of ten teeth were lost when rhBMP-7 was not applied. In the presence of rhBMP-7, eruption of teeth in group 2 was significantly improved (difference [median]: 5 mm, P<0.05, n=6). Even though there was a tendency towards increased deposition rates of cementum under rhBMP-7, this difference was not significant (Wilcoxon: P>0.05, ANOVA: P=0.002; n=6/group). In conclusion, rhBMP-7 improved survival rates and eruption of replanted teeth in growing individuals. No adverse effects were seen. Based on the present results, future clinical trials appear to be warranted.     

TRAF2 is essential for TNF-alpha-induced osteoclastogenesis.

TRAF2-deficient mice show embryonic lethality, and we developed a new in vitro differentiation system to show the function of TRAF2 in osteoclastogenesis, in which osteoclast progenitors are derived from the fetal liver of TRAF2-deficient mice. Using this system, we showed that TRAF2 is required for TNF-alpha-induced osteoclastogenesis. INTRODUCTION: TNF receptor-associated factor 2 (TRAF2) is a signal transducer for RANK and for two TNF receptor isotypes, TNFR1 and TNFR2. Because TRAF2-deficient mice show embryonic lethality, it has remained unclear whether TRAF2 is crucial in RANKL- or TNF-alpha-induced osteoclastogenesis. MATERIALS AND METHODS: Osteoclast progenitors derived from fetal liver were cultured in the presence of monocyte macrophage colony-stimulating factor (M-CSF), and flow cytometry for characterization of surface markers on these cells was performed. To examine the involvement of TRAF2 in osteoclast differentiation, we cultured osteoclast progenitors from TRAF2-deficient and wildtype mice with soluble RANKL or TNF-alpha in the presence of M-CSF, and counted the number of TRACP(+) multinucleate cells formed. c-jun N-terminal kinase (JNK) and NF-kappaB activation in osteoclast progenitors was examined by Western blot analysis and electrophoretic mobility shift assay, respectively. Nuclear factor of activated T cells (NFATc1) expression and activation were analyzed by RT-PCR and immunofluorescence staining, respectively. To examine whether TRAF2 overexpression induced osteoclastogenesis, TRAF2 was overexpressed in osteoclast progenitors form wildtype bone marrow by retrovirus infection. RESULTS AND CONCLUSIONS: Osteoclast progenitors from normal fetal liver, which were cultured with M-CSF, expressed surface molecules c-fms, Mac-1, and RANK, and could differentiate into TRACP(+) multinucleate cells in the presence of soluble RANKL or TNF-alpha. RANKL-induced osteoclastogenesis gave a reduction of 20% in the progenitors from TRAF2-deficient mice compared with that of the cells from littermate wildtype mice, whereas TNF-alpha-induced osteoclastogenesis was severely impaired in the cells from the TRAF2-deficient mice. Only a few TRACP(+) multinucleate cells were formed, and TNF-alpha-mediated activation of JNK, NF-kappaB, and NFATc1 was defective. TRAF2 overexpression induced differentiation of osteoclast progenitors from wildtype mice into TRACP(+) multinucleate cells. These results suggest that TRAF2 plays an important role in TNF-alpha-induced osteoclastogenesis.     

碳酸氢盐在小鼠精子体外获能和顶体反应中的作用

检验了精子获能和透明带（ＺＰ）及孕酮激发顶体反应是否需要ＨＣＯ３／ＣＯ２。小鼠精子分别在改良的Ｔｙｒｏｄｅ（ｍＴ－Ｂ２５，含２５ｍｍｏｌ／ＬＨＣＯ３／ＣＯ２）或ｍＴ－Ｈ（无ＨＣＯ３Ｈ／ＣＯ２，含２０ｍｍｏｌ／ＬＨｅｐｅｓ）中预培养９０ｍｉｎ后，以２步７０％和３５％ｐｅｒｃｏｌｌ／ｍＴ－Ｈ洗涤精子，并将精子重新悬浮于ｍＴ－Ｂ２５，ｍＴ－Ｂ１５ｍＴ－ＢＨ和ｍＴ－Ｈ中，用ＩＺＰ／μｌ或１５μｍｏｌ／Ｌ孕酮激发精子顶体反应。在上述ｍＴ诸培养基中，“Ｂ”精子（以ＣＴＣ荧光染色法确定）发生率为６１％～６７％；顶体反应均可达到４１．０士１．４％～４８．０±１．４％。表示精子一旦在ＨＣＯ３／Ｃ０２环境中获能，顶体反应就可在无ＨＣＯ３／ＣＯ２环境中发生。然而，精子预先在ｍＴ－Ｈ中培养，与上述相同方法处理精子，“Ｂ”型精子（２２％～３３％）明显低于前者，对ＺＰ或孕酮的刺激不起反应（１４．Ｏ士３．６～２４．７士０．６％），甚至将精子重新悬浮于ｍＴ－Ｂ２５中也不发生反应（２２．０±９．５％），表示这些精子并未获能。上述结果表明小鼠精子获能依赖于ＨＣＯ３／ＣＯ２，但顶体反应则否。     

Clusterin is not essential for androgen-regulated involution and regeneration of the normal mouse prostate

BACKGROUND: Inhibition of clusterin expression has been shown to enhance the sensitivity of prostate cancer cells to chemo and hormone therapy. Clusterin antisense oligonucleotides (ASOs) are currently in phase II human clinical trials for treatment of hormone refractory prostate cancer. However, the role of clusterin in androgen-regulated involution and regeneration of the normal prostate gland has not been established. METHODS: Prostate involution and regeneration was examined in clusterin-deficient mice undergoing up to three cycles of androgen withdrawal and restoration. RESULTS: Surprisingly, clusterin deficiency did not affect the apoptotic index, and the temporal biochemical and morphological changes associated with involution and regeneration of the normal adult prostate following multiple rounds of androgen withdrawal and replacement. CONCLUSION: Clusterin is not critical for normal prostate development or androgen-regulated involution and regrowth of the mouse prostate gland, suggesting that clusterin may have distinct functions in malignant versus normal prostatic epithelial cells.     

Cbp recruitment of Csk into lipid rafts is critical to c‐Src kinase activity and bone resorption in osteoclasts

A tyrosine kinase, c‐Src, that plays an indispensable role in ruffled border formation and bone resorption is constitutively active in osteoclasts. However, to date, the molecular mechanism underlying increased c‐Src activity in osteoclasts is unknown. To address this, we first examined the expression levels and subcellular localization of Csk, a negative regulatory kinase for c‐Src. We found that the expression level of Csk in osteoclasts was comparable with that of other tissues. However, in osteoclasts, Csk was hardly localized in lipid rafts, where c‐Src is highly expressed. Interestingly, expression of Cbp, which recruits Csk into lipid rafts through physical interaction with Csk, was very low in osteoclasts compared with other tissues. To understand the importance of Cbp in osteoclasts, we introduced Cbp into osteoclasts using an adenovirus gene delivery system. Introduction of Cbp stimulated recruitment of Csk into lipid rafts and suppressed c‐Src activity in a dose‐dependent manner. Furthermore, introduction of Cbp markedly inhibited formation of actin rings and bone‐resorbing activity in osteoclasts. In addition, treatment with RANKL and overexpression of TRAF6 or NFAT2 inhibited Cbp expression in the osteoclastogenic cell line RAW264.7 along with osteoclastic differentiation. NFAT2 overexpression also inhibited Cbp expression in spleen macrophages. Collectively, our results indicate that reduction in Cbp expression is responsible for maintaining high c‐Src activity in osteoclasts. These findings contribute to an understanding of the unique regulatory system for c‐Src in osteoclasts. © 2010 American Society for Bone and Mineral Research