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This paper provides an extended level set (X-LS) based topology optimiza- tion method for multi material design. In the proposed method, each zero level set of a level set function {\phi}ij represents the boundary between materials i and j. Each increase or decrease of {\phi}ij corresponds to a material change between the two materials. This approach reduces the dependence of the initial configuration in the optimization calculation and simplifies the sensitivity analysis. First, the topology optimization problem is formulated in the X-LS representation. Next, the reaction-diffusion equation that updates the level set function is introduced, and an optimization algorithm that solves the equilibrium equations and the reaction-diffusion equation using the fi- nite element method is constructed. Finally, the validity and utility of the proposed topology optimization method are confirmed using two- and three- dimensional numerical examples., Comment: 54 pages

The bone is the main storage site for Ca2+ and Mg2+ ions in the mammalian body. Although investigations into Ca2+ signaling have progressed rapidly and led to better understanding of bone biology, the Mg2+ signaling pathway and associated molecules remain to be elucidated. Here, we investigated the role of a potential Mg2+ signaling-related lysosomal molecule, two-pore channel subtype 2 (TPC2), in osteoclast differentiation and bone remodeling. Previously, we found that under normal Mg2+ conditions, TPC2 promotes osteoclastogenesis. We observed that under low-Mg2+ conditions, TPC2 inhibited, rather than promoted, the osteoclast differentiation and that the phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) signaling pathway played a role in the TPC2 activation under low-Mg2+ conditions. Furthermore, PI(3,5)P2 depolarized the membrane potential by increasing the intracellular Na+ levels. To investigate how membrane depolarization affects osteoclast differentiation, we generated a light-sensitive cell line and developed a system for the light-stimulated depolarization of the membrane potential. The light-induced depolarization inhibited the osteoclast differentiation. We then tested the effect of myo-inositol supplementation, which increased the PI(3,5)P2 levels in mice fed a low-Mg2+ diet. The myo-inositol supplementation rescued the low-Mg2+ diet–induced trabecular bone loss, which was accompanied by the inhibition of osteoclastogenesis. These results indicate that low-Mg2+–induced osteoclastogenesis involves changes in the role of TPC2, which are mediated through the PI(3,5)P2 pathway. Our findings also suggest that myo-inositol consumption might provide beneficial effects in Mg2+ deficiency–induced skeletal diseases.

// Kosuke Watari 1, * , Ayumi Nishitani 1, * , Tomohiro Shibata 1 , Masaki Noda 1 , Akihiko Kawahara 2 , Jun Akiba 3 , Yuichi Murakami 1, 4 , Hirohisa Yano 3 , Michihiko Kuwano 4 , Mayumi Ono 1 1 Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan 2 Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan 3 Department of Pathology, Kurume University School of Medicine, Kurume, Japan 4 Cancer Translational Research Center, St. Mary’s Institute of Health Sciences, Kurume, Japan * These authors contributed equally to this work Correspondence to: Mayumi Ono, email: mono@phar.kyushu-u.ac.jp Keywords: mTOR Ser2481, mTORC1, Raptor, rapalogs, hepatocellular carcinoma Received: February 15, 2016 Accepted: June 07, 2016 Published: June 18, 2016 ABSTRACT Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Although recent studies facilitate the identification of crucial genes and relevant regulatory pathways, therapeutic approaches against advanced HCC are insufficiently effective. Therefore, we aimed here to develop potent therapeutics to provide a reliable biomarker for the therapeutic efficacy in patients with HCC. To this end, we first compared the cytotoxic effects of various anti-cancer drugs between well differentiated (HAK-1A) and poorly differentiated (HAK-1B) cell lines established from a single HCC tumor. Of various drug screened, HAK-1B cells were more sensitive by a factor of 2,000 to the mTORC1 inhibitors (rapalogs), rapamycin and everolimus, than HAK-1A cells. Although rapalogs inhibited phosphorylation of mTOR Ser2448 in HAK-1A and HAK-1B cells, phosphorylation of mTOR Ser2481 was specifically inhibited only in HAK-1B cells. Silencing of Raptor induced apoptosis and inhibited the growth of only HAK-1B cells. Further, three other cell lines established independently from the tumors of three patients with HCC were also approximately 2,000-fold times more sensitive to rapamycin, which correlated closely with the inhibition of mTOR Ser2481 phosphorylation by rapamycin. Treatment with everolimus markedly inhibited the growth of tumors induced by poorly differentiated HAK-1B and KYN-2 cells and phosphorylation of mTOR Ser2481 in vivo . To our knowledge, this is the first study showing that the phosphorylation of mTOR Ser2481 is selectively inhibited by rapalogs in mTORC1-addicted HCC cells and may be a potential reliable biomarker for the therapeutic efficacy of rapalogs for treating HCC patients.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00441-015-2345-y) contains supplementary material, which is available to authorized users.

In mammals, the ovarian follicles are regulated at least in part by bone morphogenetic protein (BMP) family members. Dullard (also known as Ctdnep1) gene encodes a phosphatase that suppresses BMP signaling by inactivating or degrading BMP receptors. Here we report that the Col1a1-Cre-induced Dullard mutant mice displayed hemorrhagic ovarian cysts, with red blood cells accumulated in the follicles, resulting in infertility. Cells expressing Cre driven by Col1a1 2.3-kb promoter and their descendants were found in granulosa cells in the ovary and in Sertoli cells in the testis. DullardmRNA was localized to granulosa cells in the ovary. Genes involved in steroid hormone genesis including Cyp11a1, Hsd3b1 and Star were reduced, whereas expression of Smad6 and Smad7, BMP-inducible inhibitory Smads, was up-regulated in the Dullard mutant ovaries. Tamoxifen-inducible Dullard deletion in the whole body using Rosa26-CreER mice also resulted in hemorrhagic ovarian cysts in 2 weeks, which was rescued by administration of LDN-193189, a chemical inhibitor of BMP receptor kinase, suggesting that the hemorrhage in the Dullard-deficient ovarian follicles might be caused by increased BMP signaling. Thus, we conclude that Dullard is essential for ovarian homeostasis at least in part via suppression of BMP signaling.

International audience; Cells express several G-protein-coupled receptors (GPCRs) at their surfaces, transmitting simultaneous extracellular hormonal and chemical signals into cells. A comprehensive understanding of mechanisms underlying the integrated signaling response induced by distinct GPCRs is thus required. Here we found that the b2-adrenergic receptor, which induces a short cAMP response, prolongs nuclear cAMP and protein kinase A (PKA) activation by promoting endosomal cAMP production in parathyroid hormone (PTH) receptor signaling through the stimulatory action of G protein G beta gamma subunits on adenylate cyclase type 2.

Using pharmacological activation and genetic ablation of β2-adrenergic receptors (β2ARs) in mice, Nakai et al. show that β2ARs expressed on lymphocytes can regulate egress of these cells from lymph nodes, while altering the responsiveness of chemokine receptors CCR7 and CXCR4. They identify that β2ARs can physically interact with these chemokine receptors. And, in mouse models of T cell–mediated inflammation, β2AR-mediated signals are shown to inhibit trafficking of antigen-primed T cells, reducing their numbers in inflamed peripheral tissues., Lymphocyte recirculation through secondary lymphoid organs is essential for immunosurveillance and lymphocyte effector functions. Here, we show that signals through β2-adrenergic receptors (β2ARs) expressed on lymphocytes are involved in the control of lymphocyte dynamics by altering the responsiveness of chemoattractant receptors. Agonist stimulation of lymphocyte β2ARs inhibited egress of lymphocytes from lymph nodes (LNs) and rapidly produced lymphopenia in mice. Physiological inputs from adrenergic nerves contributed to retention of lymphocytes within LNs and homeostasis of their distribution among lymphoid tissues. β2ARs physically interacted with CCR7 and CXCR4, chemokine receptors promoting lymphocyte retention in LNs. Activation of β2ARs enhanced retention-promoting signals through CCR7 and CXCR4, and consequently inhibited lymphocyte egress from LNs. In models of T cell–mediated inflammatory diseases, β2AR-mediated signals inhibited LN egress of antigen-primed T cells and reduced their recruitment into peripheral tissues. Thus, this study reveals a novel mechanism for controlling lymphocyte trafficking and provides additional insights into immune regulation by the nervous system.

Profilin 1 (Pfn1), a regulator of actin polymerization, controls cell movement in a context-dependent manner. Pfn1 supports the locomotion of most adherent cells by assisting actin-filament elongation, as has been shown in skeletal progenitor cells in our previous study. However, because Pfn1 has also been known to inhibit migration of certain cells, including T cells, by suppressing branched-end elongation of actin filaments, we hypothesized that its roles in osteoclasts may be different from that of osteoblasts. By investigating the osteoclasts in culture, we first verified that Pfn1-knockdown (KD) enhances bone resorption in preosteoclastic RAW264.7 cells, despite having a comparable number and size of osteoclasts. Pfn1-KD in bone marrow cells showed similar results. Mechanistically, Pfn1-KD osteoclasts appeared more mobile than in controls. In vivo, the osteoclast-specific conditional Pfn1-deficient mice (Pfn1-cKO) by CathepsinK-Cre driver demonstrated postnatal skeletal phenotype, including dwarfism, craniofacial deformities, and long-bone metaphyseal osteolytic expansion, by 8 weeks of age. Metaphyseal and diaphyseal femurs were drastically expanded with suppressed trabecular bone mass as indicated by μCT analysis. Histologically, TRAP-positive osteoclasts were increased at endosteal metaphysis to diaphysis of Pfn1-cKO mice. The enhanced movement of Pfn1-cKO osteoclasts in culture was associated with a slight increase in cell size and podosome belt length, as well as an increase in bone-resorbing activity. Our study, for the first time, demonstrated that Pfn1 has critical roles in inhibiting osteoclast motility and bone resorption, thereby contributing to essential roles in postnatal skeletal homeostasis. Our study also provides novel insight into understanding skeletal deformities in human disorders.

A 2.5 Gbit/s burst-mode receiver for passive optical network (PON) systems with both a fast receiver settling time of 24.1 ns and a high tolerance to consecutive identical digits (CIDs) of 629 bits, by using a preamplifier with a unique peak-detecting automatic gain control function is developed. It is broadly applicable to the time- and wavelength-division multiplexed-PON systems of NG-PON2, which need a wide dynamic range suitable for a 64-way split and a transmission distance exceeding 40 km.

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 osteoclastsin vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment.In vitrostudies 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 vivoandin vitro. F-ALN was taken into bone-forming cells bothin vivoandin vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiationin vitroand it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formationin 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.

Suzuki et al. show that neural inputs to β2-adrenergic receptors expressed on lymphocytes generate the diurnal variation in the frequency of lymphocyte egress from lymph nodes, which is reflected in the magnitude of the adaptive immune response., Various aspects of the immune system display circadian rhythms. Although lymphocyte trafficking has been suggested to show diurnal variations, the mechanisms and influences on immune responses are unclear. Here, we show in mice that inputs from adrenergic nerves contribute to the diurnal variation of lymphocyte recirculation through lymph nodes (LNs), which is reflected in the magnitude of the adaptive immune response. Neural inputs to β2-adrenergic receptors (β2ARs) expressed on lymphocytes reduced the frequency of lymphocyte egress from LNs at night, which was accompanied by an increase of lymphocyte numbers in LNs. Immunization during the period of lymphocyte accumulation in LNs enhanced antibody responses. The diurnal variation of the humoral immune response was dependent on β2AR-mediated neural signals and was diminished when lymphocyte recirculation through LNs was stopped. This study reveals the physiological role of adrenergic control of lymphocyte trafficking in adaptive immunity and establishes a novel mechanism that generates diurnal rhythmicity in the immune system.

Osteoclast differentiation is one of the critical steps that control bone mass levels in osteoporosis, but the molecules involved in osteoclastogenesis are still incompletely understood. Here, we show that two-pore channel 2 (TPC2) is expressed in osteoclast precursor cells, and its knockdown (TPC2-KD) in these cells suppressed RANKL-induced key events including multinucleation, enhancement of tartrate-resistant acid phosphatase (TRAP) activities, and TRAP mRNA expression levels. With respect to intracellular signaling, TPC2-KD reduced the levels of the RANKL-induced dynamic waving of Ca(2+) in RAW cells. The search for the target of TPC2 identified that nuclear localization of NFATc1 is retarded in TPC2-KD cells. Finally, TPC2-KD suppressed osteoclastic pit formation in cultures. We conclude that TPC2 is a novel critical molecule for osteoclastogenesis.

Bone is maintained by continuous bone formation by osteoblasts provided by proliferation and differentiation of osteoprogenitors. Parathyroid hormone (PTH) activates bone formation, but because of the complexity of cells in the osteoblast lineage, how these osteoprogenitors are regulated by PTH in vivo is incompletely understood. To elucidate how signals by PTH in differentiated osteoblasts regulate osteoprogenitors in vivo, we conducted bone marrow ablation using Col1a1-constitutively active PTH/PTHrP receptor (caPPR) transgenic mice. These mice express caPPR specifically in osteoblasts by using 2.3 kb Col1a1 promoter and showed higher trabecular bone volume under steady-state conditions. In contrast, after bone marrow ablation, stromal cells recruited from bone surface extensively proliferated in the marrow cavity in transgenic mice, compared to limited proliferation in wild-type mice. Whereas de novo bone formation was restricted to the ablated area in wild-type mice, the entire marrow cavity, including not only ablated area but also outside the ablated area, was filled with newly formed bone in transgenic mice. Bone mineral density was significantly increased after ablation in transgenic mice. Bone marrow cell culture in osteogenic medium revealed that alkaline phosphatase-positive area was markedly increased in the cells obtained from transgenic mice. Furthermore, mRNA expression of Wnt-signaling molecules such as LRP5, Wnt7b, and Wnt10b were upregulated after marrow ablation in bone marrow cells of transgenic mice. These results indicate that constitutive activation of PTH/PTHrP receptor in differentiated osteoblasts enhances bone marrow ablation-induced recruitment, proliferation, and differentiation of osteoprogenitors.

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.

Parathyroid hormone/parathyroid hormone-related protein receptor (PPR) signaling is known to be involved in tooth development. In bone, extracellular matrix protein osteopontin (OPN) is a negative regulator of PPR signaling in bone formation. However, the role of OPN in modulation of PPR action in tooth development is not understood. Therefore, we examined the tooth in double mutant mice. Constitutively active PPR was expressed specifically in the odontoblasts and osteoblasts (caPPR-tg) in the presence or absence of OPN. Radiographic analysis indicated that the length of the third molar (M3) and the incisor was decreased in the caPPR-tg mice compared to wild type, and such reduction in molar and incisor length was further enhanced in the absence of OPN (caPPR-tg OPN-KO). With respect to histology of incisors, caPPR-tg induced high cellularity and irregularity in odontoblastic shape and this was enhanced by the absence of OPN. These morphological observations suggest that OPN modulates PPR signaling that are involved in tooth formation.

Objective Human synovium–derived mesenchymal stem cells (MSCs) can efficiently differentiate into mature chondrocytes. It has been suggested that DNA methylation is one mechanism that regulates human chondrogenesis; however, the methylation status of genes related to chondrogenic differentiation is not known. The purpose of this study was to investigate the CpG methylation status in human synovium–derived MSCs during experimental chondrogenesis, with a view toward potential therapeutic use in osteoarthritis. Methods Human synovium–derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed by bisulfite sequencing before and after differentiation. The expression levels of these genes were analyzed by real-time reverse transcription–polymerase chain reaction. Methylation status was also examined in human articular cartilage. Results Bisulfite sequencing analysis indicated that 10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture. Conclusion The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium–derived MSCs.

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.

Dendritic cells (DCs) are professional APCs that can control immune responses against self and altered self, typically foreign, determinants. DCs can be divided into several subsets, including CD8α+ and CD8α− DCs. These subsets possess specific functions. For example, mouse splenic CD8α+, but not CD8α− DCs selectively take up dying cells and cross-present cell-associated Ags to naive T cells. In this study, we identified genes that were more expressed in CD8α+ than CD8α− DCs by microarray analysis. Only one of these genes, when the extracellular domains were linked to human IgG Fc domain, could bind to late apoptotic or necrotic cells. This gene was a new member of the triggering receptor expressed on myeloid cells (Trem) family, Trem-like 4 (Treml4). Treml4 mRNA and protein, the latter detected with a new mAb, were predominantly expressed in spleen. Treml4, like other Trem family members, could associate with the adaptor molecule DNAX activation protein 12 kDa, but neither DNAX activation protein 10 kDa nor FcRγ. Consistent with the microarray data, we confirmed that Treml4 protein was more expressed on CD8α+ than CD8α− DCs, and we also found that Treml4 was expressed at high levels on splenic macrophages in spleen, particularly red pulp and marginal metallophilic macrophages. In addition, Treml4 expression on DCs was not changed after maturation induced by TLR ligands. Thus, Treml4 is a new Trem family molecule that is abundantly expressed on CD8α+ DCs and subsets of splenic resident macrophages, and can recognize dead cells by different types of phagocytes in spleen.

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.

Mice lacking tob2, a member of the antiproliferative family genes, had decreased bone mass, and the number of osteoclasts differentiated from bone marrow cells was increased. Overexpression of Tob2 in stromal cells repressed vitamin D3-induced osteoclasts formation. Furthermore, expression of RANKL mRNA in stromal cells was increased in the absence of Tob2 and decreased in the presence of Tob2. Tob2 interacted with vitamin D3 receptor (VDR), which suggests its involvement in vitamin D3 receptor-mediated regulation of transcription. Because VDR regulates RANKL expression, our data suggest that Tob2 negatively regulates formation of osteoclasts by suppressing RANKL expression through its interaction with VDR.

p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of a Src homology 3 (SH3) domain, a substrate domain (SD) and a Src binding domain (SBD). The SH3 domain of Cas associates with focal adhesion kinase (FAK), but its role in cellular function has not fully been understood. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking exon 2, which encodes the SH3 domain (Cas Δexon 2). In comparison to wild-type cells, Cas exon 2Δ/Δ cells showed reduced motility, which could be due to impaired tyrosine-phosphorylation of FAK and Cas, reduced FAK/Cas/Src/CrkII binding, and also impaired localization of Cas Δexon 2 to focal adhesions on fibronectin. In addition, to analyze downstream signaling pathways regulated by Cas exon 2, we performed microarray analyses. Interestingly, we found that a deficiency of Cas exon 2 up-regulated expression of CXC Chemokine Receptor-4 and CC Chemokine Receptor-5, which may be regulated by IκBα phosphorylation. These results indicate that the SH3-encoding exon of Cas participates in cell motility, tyrosine-phosphorylation of FAK and Cas, FAK/Cas/Src/CrkII complex formation, recruitment of Cas to focal adhesions and regulation of cell motility-associated gene expression in primary fibroblasts.

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

Postmenopausal osteoporosis and rheumatoid joint destruction result from increased osteoclast formation and bone resorption induced by receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor (TNF). Osteoclast formation induced by these cytokines requires NF-kappaB p50 and p52, c-Fos, and NFATc1 expression in osteoclast precursors. c-Fos induces NFATc1, but the relationship between NF-kappaB and these other transcription factors in osteoclastogenesis remains poorly understood. We report that RANKL and TNF can induce osteoclast formation directly from NF-kappaB p50/p52 double knockout (dKO) osteoclast precursors when either c-Fos or NFATc1 is expressed. RANKL- or TNF-induced c-Fos up-regulation and activation are abolished in dKO cells and in wild-type cells treated with an NF-kappaB inhibitor. c-Fos expression requires concomitant RANKL or TNF treatment to induce NFATc1 activation in the dKO cells. Furthermore, c-Fos expression increases the number and resorptive capacity of wild-type osteoclasts induced by TNF in vitro. We conclude that NF-kappaB controls early osteoclast differentiation from precursors induced directly by RANKL and TNF, leading to activation of c-Fos followed by NFATc1. Inhibition of NF-kappaB should prevent RANKL- and TNF-induced bone resorption.

An accurate assessment of the cervical lymph node metastasis status in oral cavity cancer not only helps predict the prognosis of patients, but also helps surgeons to perform the appropriate treatment. We investigated the utilization of microarray technology focusing on the differences in gene expression profiles between primary tumors of oral squamous cell carcinoma that had metastasized to cervical lymph nodes and those that had not metastasized in the hope of finding new biomarkers to serve for diagnosis and treatment of oral cavity cancer. To design this experiment, we prepared two groups: the learning case group with 30 patients and the test case group with 13 patients. All tissue samples were performed using laser captured microdissection to yield cancer cells, and RNA was isolated from purified cancer cells. To identify a predictive gene expression signature, the different gene expressions between the two groups with and without metastasis in the learning case (n = 30) were analyzed, and the 85 genes expressed differentially were selected. Subsequently, to construct a more accurate prediction model, we further selected the genes with a high power for prediction from the 85 genes using the AdaBoost algorithm. The eight candidate genes, DCTD, IL-15, THBD, GSDML, SH3GL3, PTHLH, RP5-1022P6 and C9orf46, were selected to achieve the minimum error rate. Quantitative reverse transcription-polymerase chain reaction was carried out to validate the selected genes. From these statistical methods, the prediction model was constructed including the eight genes and this model was evaluated by using the test case group. The results in 12 of 13 cases ( approximately 92.3%) were predicted correctly.

Osteoporosis is one of the most widespread and destructive bone diseases in our modern world. There is a great need for anabolic agents for bone which could reverse this disease, but few are available for clinical use. Prostaglandin E receptor (EP4) agonist (EP4A) is one of the very few anabolic agents for bone in rat, but its systemic efficacy against bone loss at sub-optimal dose is limited in mice. As osteoblasts are regulated by extracellular matrix proteins, we tested whether deficiency of osteopontin (OPN), a secreted phosphorylated protein, could modulate the effects of EP4A (ONO-AE1-329) treatment at 30 μg/kg body weight, a sub-optimal dose, for 5 days/week for 4 weeks. OPN deficiency enhanced the anabolic effects of EP4A on bone volume. Histomorphometric analysis indicated that EP4A increased mineral apposition rate as well as bone formation rate in OPN-deficient but not in wild-type mice. Neither OPN deficiency nor EP4A altered osteoclast parameters. Importantly, OPN deficiency enhanced the direct anabolic action of EP4A locally injected onto the parietal bone in inducing new bone formation. Combination of OPN deficiency and EP4A treatment caused an increase in mineralized nodule formation in the cultures of bone marrow cells. Finally, OPN deficiency enhanced anabolic action of EP4A in the mice subjected to ovariectomy. These data indicate that OPN deficiency enhances the actions of EP4A at sub-optimal dose.

Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of bone-formation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/osteoblastic migration and bone mass.

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.

Objective: Apoptosis of chondrocytes in articular cartilage has been observed in rheumatoid arthritis patients. However, molecules involved in such chondrocyte apoptosis in arthritic joints have not been fully understood. We previously observed that apoptosis of chondrocytes is enhanced in a murine arthritis model induced by injection with anti–type II collagen antibodies and lipopolysaccharide (mAbs/LPS), and osteopontin (OPN) deficiency suppresses chondrocyte apoptosis in this arthritis model in vivo. To understand how OPN deficiency renders resistance against chondrocyte apoptosis, we examined the cellular basis for this protection. Design: Chondrocytes were prepared from wild-type and OPN-deficient mouse ribs, and tumor necrosis factor (TNF)–α–induced cell death was examined based on lactate dehydrogenase (LDH) release assay and TUNEL assay. Results: TNF-α treatment induced LDH release in wild-type chondrocytes, while OPN deficiency suppressed such LDH release in the cultures of these cells. TNF-α–induced increase in the number of TUNEL-positive cells was observed in wild-type chondrocytes, while OPN deficiency in chondrocytes suppressed the TNF-α induction of TUNEL-positive cells. OPN deficiency suppressed TNF-α–induced increase in caspase-3 activity in chondrocytes in culture. Furthermore, OPN overexpression in chondrocytes enhanced TNF-α–induced apoptosis. Conclusion: These results indicated that the presence of OPN in chondrocytes is involved in the susceptibility of these cells to TNF-α–induced apoptosis.

Extracellular matrix production and degradation by bone cells are critical steps in bone metabolism. Mutations of the gene encoding MMP-2, an extracellular matrix-degrading enzyme, are associated with a human genetic disorder characterized by subcutaneous nodules, arthropathy, and focal osteolysis. It is not known how the loss of MMP-2 function results in the pathology. Here, we show that Mmp2(-/-) mice exhibited opposing bone phenotypes caused by an impaired osteocytic canalicular network. Mmp2(-/-) mice showed decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae. In the long bones, there was moderate disruption of the osteocytic networks and reduced bone density throughout life, whereas osteoblast and osteoclast function was normal. In contrast, aged but not young Mmp2(-/-) mice had calvarial sclerosis with osteocyte death. Severe disruption of the osteocytic networks preceded osteocyte loss in Mmp2(-/-) calvariae. Successful transplantation of wild-type periosteum restored the osteocytic canalicular networks in the Mmp2(-/-) calvariae, suggesting local roles of MMP-2 in determining bone phenotypes. Our results indicate that MMP-2 plays a crucial role in forming and maintaining the osteocytic canalicular network, and we propose that osteocytic network formation is a determinant of bone remodeling and mineralization.

Phosphate (Pi) plays a critical role in the maintenance of mineralized tissues and signaling in the intracellular environment. Although extracellular phosphate concentration is maintained at fixed levels, physiological machineries involved in phosphate homeostasis in bone, which is the largest phosphate storage site, have not yet been fully elucidated. Here we examined the role of osteopontin (OPN) in a high-Pi diet load-induced bone loss. A high-Pi diet significantly reduced bone mineral density as well as bone mass in wild type. In contrast, OPN deficiency totally prevented reduction in bone mineral density and bone mass. Analyses of bone turnover-related components revealed that bone formation parameters (bone formation rate and mineral apposition rate) were enhanced by high-Pi diet load similarly in wild-type and OPN-deficient mice. In sharp contrast, bone resorption parameters (osteoclast number and osteoclast surface) were enhanced by high-Pi diet load in wild type but not at all in OPN-deficient mice. Bone marrow cell cultures revealed no major effects of OPN deficiency on high-Pi diet modulation of mineralized nodule formation in culture. On the other hand, tartrate-resistant acid phosphatase-positive multinucleated cell development in cultures were enhanced by high-Pi diet load in wild-type cells, but such effects of high Pi-diet were totally abolished in the absence of OPN. These data indicated that OPN is needed for osteoclastic activity to resorb bone on high phosphate loading.

Molecular mechanisms underlying unloading-induced reduction of bone formation have not yet been fully understood. In vitro, Runx2 has been suggested to be involved in mechanical signaling in osteoblasts. However, the roles of Runx2 in vivo during the bone response to mechanical stimuli have not yet been known. The purpose of this paper was to examine the roles of Runx2 in unloading-induced bone loss in vivo. Tail suspension was conducted for 2 wk using 9- to 11-wk-old Runx2 heterozygous knockout mice (Runx2+/−) and wild-type (Wt) littermates. Bones were subjected to two-dimensional micro-x-ray computed tomography, bone histomorphometry and RT-PCR analyses. Loss of half Runx2 gene dosage-exacerbated unloading-induced bone loss in trabecular and cortical envelopes. Unloading-induced reduction in mineral apposition rate and bone formation rate in cortical bone as well as trabecular bone was exacerbated in Runx2+/− mice, compared with Wt mice. Bone resorption parameters were not significantly affected by unloading or Runx2+/− genotype. Basal Runx2 and osterix mRNA levels in bone were reduced by 50% in Wt, whereas unloading in Runx2+/− mice did not further alter Runx2 and osterix mRNA levels. In contrast, osteocalcin mRNA levels were reduced by unloading, regardless of Runx2 gene dosage. These data demonstrated that full Runx2 gene dosage is required for maintaining normal function of osteoblasts in mechanical unloading or nonphysiological condition. Finally, we propose Runx2 as a critical target gene in unloading to alter osteoblastic activity and bone formation in vivo.

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)–induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2–induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.

Bone remodelling, the mechanism by which vertebrates regulate bone mass, comprises two phases, namely resorption by osteoclasts and formation by osteoblasts; osteoblasts are multifunctional cells also controlling osteoclast differentiation. Sympathetic signalling via beta2-adrenergic receptors (Adrb2) present on osteoblasts controls bone formation downstream of leptin. Here we show, by analysing Adrb2-deficient mice, that the sympathetic nervous system favours bone resorption by increasing expression in osteoblast progenitor cells of the osteoclast differentiation factor Rankl. This sympathetic function requires phosphorylation (by protein kinase A) of ATF4, a cell-specific CREB-related transcription factor essential for osteoblast differentiation and function. That bone resorption cannot increase in gonadectomized Adrb2-deficient mice highlights the biological importance of this regulation, but also contrasts sharply with the increase in bone resorption characterizing another hypogonadic mouse with low sympathetic tone, the ob/ob mouse. This discrepancy is explained, in part, by the fact that CART ('cocaine amphetamine regulated transcript'), a neuropeptide whose expression is controlled by leptin and nearly abolished in ob/ob mice, inhibits bone resorption by modulating Rankl expression. Our study establishes that leptin-regulated neural pathways control both aspects of bone remodelling, and demonstrates that integrity of sympathetic signalling is necessary for the increase in bone resorption caused by gonadal failure.

Mice homozygous for klotho gene deletion are well established aging models as they mimic certain aspects of human senescence e.g. osteoporosis. Induced senescence may affect cellular functions and alter the histological properties of the extracellular matrices. The present study examined the histological and ultrastructural features of osteocytes and the surrounding bone matrix in klotho-deficient mice. As expected, osteoblasts showed a flattened shape with a weak immunoreactivity for alkaline phosphatase, and the bone matrix contained many empty osteocytic lacunae. The walls of both normal and empty lacunae were intensely immunopositive for osteopontin and dentin matrix protein-1, but featured an inconsistent immunoreactivity for osteocalcin and type I collagen. Not surprisingly, TUNEL-positivity, indicative of apoptosis, was found in many osteoblasts, osteocytes, and bone marrow cells of the klotho-deficient mice. In transmission electron microscopy, an amorphous matrix containing non-collagenous organic materials was recognizable around osteoblasts and in the osteocytic lacunae. Some osteoblasts on the bone surface featured these amorphous materials in vacuoles associated with their trans-Golgi network, indicating that, under klotho-deficient conditions, they synthesize and secrete the non-collagenous structures. Some osteocytes displayed pyknosis or degenerative traits. Thus, our findings provide histological evidence that klotho gene deletion influences the spatial distribution of osteocytes and the synthesis of bone matrix proteins in addition to the accelerated aging of bone cells.

Sclerostin (SOST), a member of the cystine-knot superfamily, is essential for proper skeletogenesis because a loss-of-function mutation in the SOST gene results in sclerosteosis featured with massive bone growth in humans. To understand the function of SOST in developmental skeletal tissue formation, we examined SOST gene expression in embryonic osteogenesis in vitro and in vivo. During osteoblastic differentiation in primary calvarial cells, the levels of SOST expression were increased along with those of alkaline phosphatase activity and nodule formation. In situ hybridization study revealed that SOST mRNA expression was observed in the digits in embryonic 13-d limb buds, and SOST expression was observed in osteogenic front in embryonic 16.5-d postcoitus embryonic calvariae, and this expression persisted in the peripheral area of cranial bone in the later developmental stage (embryonic 18.5-d post coitum). These temporal and spacial expression patterns in vivo and in vitro were in parallel to those of osterix (Osx), which is a critical transcriptional factor for bone formation. Similar coexpression of SOST and Osx mRNA was observed when the primary osteoblastic calvarial cells were cultured in the presence of bone morphogenetic protein (BMP)2 in vitro. Moreover, endogenous expression of SOST and Osx mRNA was inhibited by infection of noggin-expression adenovirus into the primary osteoblastic calvarial cells, suggesting that endogenous BMPs are required for these cells to express SOST and Osx mRNA. Thus, expression and regulation of SOST under the control of BMP were closely associated with those of Osx in vivo and in vitro.

Osteoblasts and chondroblasts are derived from common mesenchymal progenitors. Although bone morphogenetic protein induces mesenchymal differentiation into both osteogenic and chodrogenic lineage cells in vitro, its inhibitor, Noggin, is expressed exclusively during chondrogenic but not osteogenic differentiation in an embryonal carcinoma-derived mesodermal cell line, C1. We hypothesized that Noggin may regulate cell differentiation in a lineage-specific manner. To test this hypothesis, Noggin was overexpressed using recombinant adenovirus (Ad/Noggin) in mesodermal C1 cells to examine whether Noggin specifically inhibits chondrogenic differentiation. Noggin overexpression by recombinant adenovirus infection reduced Sox9, patched, Ihh, and type II, X, and XI collagen mRNA expression levels in C1 cell aggregates that were induced to differentiate into chondrocyte lineage by culturing in differentiation medium. In contrast, Noggin overexpression did not affect osteogenic differentiation in C1 cells because osteoblast phenotypic markers such as osteocalcin and alkaline phosphatase mRNA levels were not altered. We further examined whether Noggin also differentially affects chondrogenesis and osteogenesis in limb development by using organ cultures of long bone. Ad/Noggin infection into 15.5 d post conception limb skeletal rudiments that were cultured on filter membrane in vitro or on the chorioallantoic membranes in ovo inhibited the levels of chondrogenesis, which were evaluated based on alcian blue staining. These results suggest that Noggin specifically blocks chondrogenic differentiation, rather than osteogenic differentiation, in mesodermal stem cell line C1 and skeletal cells.

Runx2 is prerequisite for the osteoblastic differentiation in vivo. To elucidate Runx2 gene functions in adult bone metabolism, we conducted bone marrow ablation in Runx2 heterozygous knockout mice and found that aged (but not young) adult Runx2 heterozygous knockout mice have reduced new bone formation capacity after bone marrow ablation. We also found that bone marrow cells from aged Runx2 heterozygous knockout mice have reduced ALP+ colony-forming potential in vitro. This indicates that full Runx2 dosage is needed for the maintenance of osteoblastic activity in adult mice. Introduction: Null mutation of the Runx2 gene results in total loss of osteoblast differentiation, and heterozygous Runx2 deficiency causes cleidocranial dysplasia in humans and mice. However, Runx2 gene functions in adult bone metabolism are not known. We therefore examined the effects of Runx2 gene function in adult mice with heterozygous loss of the Runx2 gene. Materials and Methods: Bone marrow ablation was conducted in young adult (2.5 ± 0.5 months old) or aged adult (7.5 ± 0.5 months old) Runx2 heterozygous knockout mice and wildtype (WT) littermates. Cancellous bone regeneration was evaluated by 2D μCT. Results: Although new bone formation was observed after bone marrow ablation in the operated bone marrow cavity of WT mice, such bone formation was significantly reduced in Runx2 heterozygous knockout mice. Interestingly, this effect was observed specifically in aged but not young adult mice. Runx2 heterozygous deficiency in aged mice significantly reduced the number of alkaline phosphatase (ALP)+ cell colonies in the bone marrow cell cultures, indicating a reduction in the numbers of osteoprogenitor cells. Such effects of heterozygous Runx2 deficiency on osteoblasts in vitro was specific to the cells from aged adult mice, and it was not observed in the cultures of marrow cells from young adult mice. Conclusion: These results indicate that full gene dosage of Runx2 is required for cancellous bone formation after bone marrow ablation in adult mice.

CIZ (Cas interacting zinc finger protein), also called Nmp4 (nuclear matrix protein 4), is a nucleo-cytoplasmic shuttling transcription factor that regulates the expression of collagen and matrix metalloproteinases. CIZ/Nmp4 was originally cloned by its binding to p130(Cas), a focal adhesion protein, and was recently shown to suppress BMP2 (bone mophogenetic protein 2) signalling. To explore the physiological role of CIZ/Nmp4, we disrupted CIZ/Nmp4-gene by inserting beta-galactosidase and neomycin resistance genes into the 2nd exon of CIZ/Nmp4-gene, which is utilized by all the sequenced alternative forms. CIZ-/- mice were born and grew to adulthood. Although they tend to be smaller than wild-type mice, no pathological abnormality was observed except in the testis. Histological analysis of the testes revealed variable degrees of spermatogenic cell degeneration within the seminiferous tubules of CIZ-/- mice, resembling the histology of the 'Germinal-cell aplasia with focal spermatogenesis'. Some of the CIZ-/- male mice developed infertility. TUNEL assay on testis sections revealed an increased occurrence of apoptosis of spermatogenic cells in the testes of CIZ-/- mice. CIZ/Nmp4 was co-localized with Smad1 in the testis, suggesting that a disregulation of BMP signalling could cause these phenotypes. These results suggest that CIZ/Nmp4 plays roles in the progress and the maintenance of spermatogenesis.

Tob (transducer of erbB2) is a member of antiproliferative family proteins and acts as a bone morphogenic protein inhibitor as well as a suppressor of proliferation in T cells, which have been implicated in postmenopausal bone loss. To determine the effect of Tob deficiency on estrogen deficiency-induced bone loss, we analyzed bone metabolism after ovariectomy or sham operation in Tob-deficient mice. Ovariectomy in WT mice decreased trabecular bone volume and bone mineral density (BMD) as expected. In Tob-deficient mice, ovariectomy reduced bone volume and BMD. However, even after ovariectomy, both trabecular bone volume and BMD levels in Tob-deficient bone were comparable to those in sham-operated WT bones. Bone formation parameters (mineral apposition rate and bone formation rate) in the ovariectomized Tob-deficient mice were significantly higher than those in the ovariectomized WT mice. In contrast, the ovariectomy-induced increase in the bone resorption parameters, osteoclast surface, and osteoclast number was similar between Tob-deficient mice and WT mice. Furthermore, in ex vivo nodule formation assay, ovariectomy-induced enhancement of nodule formation was significantly higher in the bone marrow cells from Tob-deficient mice than in the bone marrow cells from ovariectomized WT mice. Both Tob and estrogen signalings converge at bone morphogenic protein activation of alkaline phosphatase and GCCG-reporter gene expression in osteoblasts, revealing interaction between the two signals. These data indicate that Tob deficiency prevents ovariectomy-induced bone loss through the superenhancement of osteoblastic activities in bone and that this results in further augmentation in the bone formation rate and the mineral apposition rate after ovariectomy in vivo .