Noggin mRNA Research Articles

Expression of Noggin and Gremlin1 and its implications in fine-tuning BMP activities in mouse cartilage tissues.

Increasing evidence supports the idea that bone morphogenetic proteins (BMPs) regulate cartilage maintenance in the adult skeleton. The aim of this study is to obtain insight into the regulation of BMP activities in the adult skeletal system. We analyzed expression of Noggin and Gremlin1, BMP antagonists that are known to regulate embryonic skeletal development, in the adult skeletal system by Noggin-LacZ and Gremlin1-LacZ knockin reporter mouse lines. Both reporters are expressed in the adult skeleton in a largely overlapping manner with some distinct patterns. Both are detected in the articular cartilage, pubic symphysis, facet joint in the vertebrae, and intervertebral disk, suggesting that they regulate BMP activities in these tissues. In a surgically induced knee osteoarthritis model in mice, expression of Noggin mRNA was lost from the articular cartilage, which correlated with loss of BMP2/4 and pSMAD1/5/8, an indicator of active BMP signaling. Both reporters are also expressed in the sterna and rib cartilage, suggesting an extensive role of BMP antagonism in adult cartilage tissue. Moreover, Noggin-LacZ was detected in sutures in the skull and broadly in the nasal cartilage, while Gremlin1-LacZ exhibits a weaker and more restricted expression domain in the nasal cartilage. These results suggest broad regulation of BMP activities by Noggin and Gremlin1 in cartilage tissues in the adult skeleton, and that BMP signaling and its antagonism by NOGGIN play a role in osteoarthritis development. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1671-1682, 2017.

Intrathecal Noggin administration in rats temporally ameliorates mechanical allodynia induced by a chronic constriction injury.

Chronic intractable neuropathic pain after central or peripheral nervous system injury remains refractory to therapeutic intervention. Using microarray and RT-qPCR methods, we found that Noggin mRNA is downregulated in the lumbar enlargement 2 weeks after chronic constriction injury (CCI) in rats.Eight-week-old female Sprague Dawley rats were used for the CCI model. Two weeks after CCI, rats underwent a laminectomy at L5 under halothane anesthesia, and a silicone tube connected to an osmotic minipump was inserted intrathecally for 14 days. Rats were administered Noggin ranging from 10 ng/ml to 10 μg/ml. Phosphate buffered saline (PBS) was used as a control. The time course of mechanical allodynia was assessed for 5 weeks using von Frey filaments. An ANOVA showed that rats administered Noggin at 2 μg/ml had significantly less mechanical allodynia compared with controls.We next compared the effect of intrathecal administration (14 days) of Noggin (2 μg/ml), bone morphogenetic protein 4 (BMP4; 2 μg/ml), or BMP4 (μg/ml) + Noggin (μg/ml) with controls. Only Noggin administration significantly reduced mechanical allodynia in the CCI model.Fluorescence immunohistochemistry indicated that Noggin administration decreased astrocyte accumulation in the dorsal horn compared with PBS after administration for one week. BMP4-driven conversion of oligodendrocyte progenitor cells (OPCs) to type 2 astrocytes is inhibited by Noggin Hampton et al. (2007) . We speculated that Noggin administration inhibits the conversion of OPCs to astrocytes, and decreases glial fibrillar acidic protein expression. This histological condition could decrease neuropathic pain.

Surface-mediated delivery of siRNA from fibrin hydrogels for knockdown of the BMP-2 binding antagonist noggin

Antagonists and inhibitory molecules responsible for maintaining tissue homeostasis can present a significant barrier to healing when tissue engineering/regenerative medicine strategies are employed. One example of this situation is the up-regulation of antagonists such as noggin in response to increasing concentrations of bone morphogenetic protein-2 (BMP-2) present from endogenous bone repair processes or delivered exogenously from biomaterials (synthetic bone grafts). While recombinant human (rh)BMP-2 delivered from synthetic bone grafts has been shown to be an effective alternative to autografts and allografts, the supraphysiological doses of rhBMP-2 have led to clinically-adverse side effects. The high rhBMP-2 dosage may be required, in part, to overcome the presence of antagonists such as noggin. Small interfering RNA (siRNA) is an appealing approach to overcome this problem because it can knock-down antagonists or inhibitory molecules in a temporary manner. Here, we conducted fundamental studies on the delivery of siRNA from material surfaces as a means to knock-down antagonists like noggin. Non-viral cationic lipid (Lipofectamine)–siRNA complexes were delivered from a fibrin hydrogel surface to MC3T3-E1 preosteoblasts that were treated with a supraphysiological dose of rhBMP-2 to achieve noggin mRNA expression levels higher than cells naïve to rhBMP-2. Confocal microscopy and flow cytometry showed intracellular uptake of siRNA in over 98% of MC3T3-E1 cells after 48h. Doses of 0.5 and 1μg noggin siRNA were able to significantly reduce noggin mRNA to levels equivalent to those in MC3T3-E1 cells not exposed to rhBMP-2 with no effects on cell viability. Statement of significanceSmall interfering RNA (siRNA) has been considered for treatment of diseases ranging from Alzheimer’s to cancer. However, the ability to use siRNA in conjunction with biomaterials to direct tissue regeneration processes has received relatively little attention. Using the bone morphogenetic protein 2 antagonist, noggin, as a model, this research describes an approach to knock-down molecules that are inhibitory to desired regenerative pathways at the mRNA level via siRNA delivery from a hydrogel surface. Interactions between the material (fibrin) surface and polycation–siRNA complexes, release of the siRNA from the material surface, high levels of cellular uptake/internalization of siRNA, and significant knockdown of the targeting (noggin) mRNA are demonstrated. Broader future applications include those to nerve regeneration, cardiovascular tissue engineering, directing (stem) cell behavior, and mitigating inflammatory responses to materials.

Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity.

In vertebrates, smooth muscle cells (SMCs) can reversibly switch between contractile and proliferative phenotypes. This involves various molecular mechanisms to reactivate developmental signaling pathways and induce cell dedifferentiation. The protein RBPMS2 regulates early development and plasticity of digestive SMCs by inhibiting the bone morphogenetic protein pathway through its interaction with NOGGIN mRNA. RBPMS2 contains only one RNA recognition motif (RRM) while this motif is often repeated in tandem or associated with other functional domains in RRM-containing proteins. Herein, we show using an extensive combination of structure/function analyses that RBPMS2 homodimerizes through a particular sequence motif (D-x-K-x-R-E-L-Y-L-L-F: residues 39–51) located in its RRM domain. We also show that this specific motif is conserved among its homologs and paralogs in vertebrates and in its insect and worm orthologs (CPO and MEC-8, respectively) suggesting a conserved molecular mechanism of action. Inhibition of the dimerization process through targeting a conserved leucine inside of this motif abolishes the capacity of RBPMS2 to interact with the translational elongation eEF2 protein, to upregulate NOGGIN mRNA in vivo and to drive SMC dedifferentiation. Our study demonstrates that RBPMS2 possesses an RRM domain harboring both RNA-binding and protein-binding properties and that the newly identified RRM-homodimerization motif is crucial for the function of RBPMS2 at the cell and tissue levels.

Effects of electro-acupuncture on learning and memory and the expression of Noggin mRNA in the hippocampus of rats with chronic cerebral ischemia

Objective To observe the effects of electro-acupuncture (EA) on learning,memory and the expression of Noggin mRNA in the hippocampus after cerebral ischemia.Methods One hundred and four male Sprague-Dawley (SD) rats were subjected to a modified form of permanent bilateral carotid artery ligation to produce a model of chronic cerebral ischemia.They were then randomly divided into a model group and an EA group,with 52 rats in each group.Each group was then subdivided into 1,2,4 and 6 weeks subgroups,with 13 rats in each subgroup.The EA group received EA administered to the Baihui (DU20) and Dazhui (BU14) acu-points; the model group received regular feeding only.The Morris water maze test was administered in both groups at 5 days before the rats were sacrificed.At the 1,2,4 and 6 week time points,6 rats were selected from each subgroup randomly and the expression of Noggin mRNA in the hippocampus was detected using RT-PCR techniques.Results The Morris water maze test showed significantly lower escape latency in the EA group at 2,4 and 6 weeks.Swimming time in the first quadrant was also significantly different at all time points.The average level of Noggin mRNA in the EA group was significantly higher than that in the model group at all of the time points.After 6 weeks,the average level of Noggin mRNA in the EA group was significantly lower than that at the other time points within the EA group.Conclusion EA can improve spatial learning and memory and elevate the expression of Noggin mRNA in the hippocampus in cases of chronic cerebral ischemia. Key words: Electro-acupuncture; Hippocampus; Learning; Memory

L51P — A BMP2 variant with osteoinductive activity via inhibition of Noggin

Bone morphogenetic proteins (BMP) have to be applied at high concentrations to stimulate bone healing. The limited therapeutic efficacy may be due to the local presence of BMP antagonists such as Noggin. Thus, inhibiting BMP antagonists is an attractive therapeutic option. We hypothesized that the engineered BMP2 variant L51P stimulates osteoinduction by antagonizing Noggin-mediated inhibition of BMP2. Primary murine osteoblasts (OB) were treated with L51P, BMP2, and Noggin. OB proliferation and differentiation were quantified with XTT and alkaline phosphatase (ALP) assays. BMP receptor dependent intracellular signaling in OB was evaluated with Smad and p38 MAPK phosphorylation assays. BMP2, Noggin, BMP receptor Ia/Ib/II, osteocalcin, and ALP mRNA expressions were analyzed with real-time PCR. L51P stimulated OB differentiation by blocking Noggin mediated inhibition of BMP2. L51P did not induce OB differentiation directly and did not activate BMP receptor dependent intracellular signaling via the Smad pathway. Treatment of OB cultures with BMP2 but not with L51P resulted in an increased expression of ALP, BMP2, and Noggin mRNA. By inhibiting the BMP antagonist Noggin, L51P enhances BMP2 activity and stimulates osteoinduction without exhibiting direct osteoinductive function. Indirect osteoinduction with L51P seems to be advantageous to osteoinduction with BMP2 as BMP2 stimulates the expression of Noggin thereby self-limiting its own osteoinductive activity. Treatment with L51P is the first protein-based approach available to augment BMP2 induced bone regeneration through inhibition of BMP antagonists. The described strategy may help to decrease the amounts of exogenous BMPs currently required to stimulate bone healing.

Evo-devo: Hydra raises its Noggin

Noggin, along with other secreted bone morphogenetic protein (BMP) inhibitors, plays a crucial role in neural induction and neural tube patterning as well as in somitogenesis, cardiac morphogenesis and formation of the skeleton in vertebrates. The BMP signalling pathway is one of the seven fundamental pathways that drive embryonic development and pattern formation in animals. Understanding its evolutionary origin and role in pattern formation is, therefore, important to evolutionary developmental biology (evo-devo). We have studied the evolutionary origin of BMP-Noggin antagonism in hydra, which is a powerful diploblastic model to study evolution of pattern-forming mechanisms because of the unusual cellular dynamics during its pattern formation and its remarkable ability to regenerate. We cloned and characterized the noggin gene from hydra and found it to exhibit considerable similarity with its orthologues at the amino acid level. Microinjection of hydra Noggin mRNA led to duplication of the dorsoventral axis in Xenopus embryos, demonstrating its functional conservation across the taxa. Our data, along with those of others, indicate that the evolutionarily conserved antagonism between BMP and its inhibitors predates bilateral divergence. This article reviews the various roles of Noggin in different organisms and some of our recent work on hydra Noggin in the context of evolution of developmental signalling pathways.

RNA-Binding Protein FXR2 Regulates Adult Hippocampal Neurogenesis by Reducing Noggin Expression

In adult mammalian brains, neurogenesis persists in the subventricular zone of the lateral ventricles (SVZ) and the dentate gyrus (DG) of the hippocampus. Although evidence suggest that adult neurogenesis in these two regions is subjected to differential regulation, the underlying mechanism is unclear. Here, we show that the RNA-binding protein FXR2 specifically regulates DG neurogenesis by reducing the stability of Noggin mRNA. FXR2 deficiency leads to increased Noggin expression and subsequently reduced BMP signaling, which results in increased proliferation and altered fate specification of neural stem/progenitor cells in DG. In contrast, Noggin is not regulated by FXR2 in the SVZ, because Noggin expression is restricted to the ependymal cells of the lateral ventricles, where FXR2 is not expressed. Differential regulation of SVZ and DG stem cells by FXR2 may be a key component of the mechanism that governs the different neurogenic processes in these two adult germinal zones.

Changes in Gene Expression during Pituitary Morphogenesis and Organogenesis in the Chick Embryo

The anterior pituitary gland plays an important role in the regulation of many physiological processes. Formation of Rathke's pouch (RP), the precursor of the anterior pituitary, involves evagination of the oral ectoderm in a multi-step process regulated by cell interactions, signaling pathways, and transcription factors. Chickens are an excellent model to study development because of the availability of large sample sizes, accurate timing of development, and embryo accessibility. The aim of this study was to quantify mRNA expression patterns in the developing chicken anterior pituitary to evaluate the chicken embryo as a model for mammalian pituitary development. The expression profiles of 16 genes differentially expressed in RP and neuroectoderm were determined in this study. Among these, Pitx1, Pitx2, and Hesx1 mRNA levels were high on embryonic days (e) 2.5 to e3 in RP and decreased during development. Expression of Pit1 and Tbx19 mRNA in RP reached the highest levels by e7 and e6.5, respectively. Levels of glycoprotein subunit α mRNA increased beginning at e4. FGF8 mRNA showed the highest expression at e3 to e3.5 in neuroectoderm. BMP2 showed slight decreases in mRNA expression in both tissues during development, while Isl1 and Noggin mRNA expression increased in later development. Taken together, we present the first quantitative transcriptional profile of pituitary organogenesis. Our results will help further understanding of the functional development of this gland. Moreover, because of the high similarity in gene expression patterns observed between chicken and mouse, chickens could serve as an excellent model to study genetic and molecular mechanisms underlying pituitary development.

Hypoxia inhibits hepcidin expression in HuH7 hepatoma cells via decreased SMAD4 signaling

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H(2)O(2), hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

Reduced production of noggin by immune cells of patients with relapsing–remitting multiple sclerosis

Multiple sclerosis (MS) plaques are characterized by neurodegeneration, astrogliolis, the presence of immature oligodendrocytes and infiltrating immune cells. Recent studies revealed a putative role for noggin in both neurogenesis and oligodenrocytes development. In order to study the potential of peripheral immune cells to contribute to neurogenesis in MS, we studied the mRNA expression, protein secretion and regulation profile of noggin in peripheral blood mononuclear cells (PBMCs) of untreated patients with relapsing–remitting MS (RR-MS), interferon-β (IFN-β) treated RR-MS patients compared to matched healthy controls (HC).Basal levels of noggin mRNA expression, determined by quantitative real-time PCR were lower in untreated patients than in HC. No differences were found between untreated patients and IFN-β treated patients. Similarly, the secreted levels of noggin, detected in 24h PBMCs supernatants by ELISA, were decreased in untreated RR-MS patients than in HC. Again no significant differences were found between untreated patients and IFN-β treated patients. Stimulation with anti-CD3/CD28 mAbs increased noggin mRNA expression in untreated patients but not in HC. However, noggin mRNA levels in untreated patients PBMCs stimulated with anti-CD3/CD28 did not reach noggin levels in unstimulated PBMCs of HC. Purification of monocytes (CD14+) and T cells (CD3+ cells) by magnet-activated cell separation has demonstrated that noggin mRNA is predominantly expressed in CD3+ cells in both HC and in RR-MS patients. This pattern also appeared in protein level of noggin, tested by Western blot. The incubation of the PBMCs with TNF-α increased the expression of noggin only in HC group. In conclusion, T cells possess the potential to participate in the induction of neurogeneration by the production of noggin. This potential seems to be defective in immune cells of RR-MS patients as there is reduced mRNA expression and protein secretion levels of noggin, insufficient stimulatory effect of CD3/CD28 stimulation and unresponsiveness to TNF-α in these patients PBMCs.

Bone morphogenetic protein 2 inhibits FSH responsiveness in hen granulosa cells

Prior to follicle selection into the preovulatory hierarchy, hen granulosa cells from prehierarchal follicles remain undifferentiated, as defined in part by the virtual absence of LHR mRNA expression and inability to produce progesterone. It has previously been proposed that prior to follicle selection, granulosa cells are actively maintained in an undifferentiated state by epidermal growth factor receptor ligands (EGFRL) signaling via the MAP kinase/extracellular regulated kinase pathway. Moreover, there is recent evidence that EGFRL/MAP kinase signaling modulates FSH receptor (FSHR) transcription, in part, via inhibitor of differentiation/DNA-binding (ID) proteins. In the present studies with undifferentiated granulosa, recombinant human (rh) bone morphogenetic protein 2 (BMP2) induced the phosphorylation of SMAD1/5/8, and blocked transforming growth factor β and FSH-induced FSHR expression and progesterone production. Significantly, BMP2 rapidly induced mRNAs encoding betacellulin and EGF, plus ID proteins (ID1, ID3, and ID4). Alternatively, the bioactivity of BMPs can be modulated by one or more BMP antagonists, including noggin (NOG). NOG mRNA is expressed by both hen granulosa and theca tissues from prehierarchal follicles. Pretreatment of cultured granulosa with rh NOG reversed both the stimulatory effects of BMP2 on ID1, ID3, and ID4 expression and the inhibitory effects of BMP2 on FSHR mRNA levels and progesterone production. Collectively, these data provide evidence that prior to follicle selection, BMP2 signaling contributes toward maintaining granulosa cells in an undifferentiated state. The actions of BMP2 are, at least in part, mediated indirectly via enhanced EGFRL expression and ERBB receptor-mediated MAP kinase signaling, and can be modulated by the autocrine/paracrine production of NOG.

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos

Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.

105 NOGGIN CONTRIBUTES TO THE OSTEOLYTIC RESPONSE IN BONE METASTASIS OF PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2010105 NOGGIN CONTRIBUTES TO THE OSTEOLYTIC RESPONSE IN BONE METASTASIS OF PROSTATE CANCER Chiara Secondini, Antoinette Wetterwald, Ruth Schwaninger, Marco G. Cecchini, and George N. Thalmann Chiara SecondiniChiara Secondini More articles by this author , Antoinette WetterwaldAntoinette Wetterwald More articles by this author , Ruth SchwaningerRuth Schwaninger More articles by this author , Marco G. CecchiniMarco G. Cecchini More articles by this author , and George N. ThalmannGeorge N. Thalmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.154AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Members of the bone morphogenetic protein (BMP) and the wingless (Wnt) protein family play a relevant role in the osteoblast response to prostate cancer (CaP) bone metastasis and their activity is modulated by extracellular antagonists. Lack of expression of the BMP antagonist noggin by CaP cells determines their osteoinductive activity in vivo. Osteolytic cells express noggin constitutively. The Wnt-antagonist dickkopf-1 (DKK-1) represses bone formation and noggin may act in analogy on BMP activity. We utilized a RNA silencing strategy to investigate whether constitutive noggin expression by an osteolytic CaP cell line determines the osteolytic phenotype. METHODS Luciferase-expressing PC-3 cell clones were transfected with shRNA vectors to produce stable noggin-knock down (Nog-KD) clones. A vector encoding a non-targeting shRNA (mock) was used as negative control. Silencing efficiency was monitored by real time PCR and immunoblotting, respectively. Nog-KD and mock clones were xenografted into the tibia of nude mice and intra-osseous growth and osteolytic effect were monitored by bioluminescent imaging (BLI) and radiography, respectively. 3-D images of the xenografts were generated by micro-tomography (£gCT). Bone structural parameters were analyzed by peripheral quantitative computerized tomography (pQCT) and histomorphometry. RESULTS Significant noggin mRNA and protein expression decrease in several Nog-KD clones with unchanged growth rates, osteolytic cytokine expression (PTHrP and IL-8), and of DKK-1 In vitro were observed. Tumor growth in bone xenografts of different mock and Nog-KD clones was only marginally or not affected. Radiographic and fÝCT analysis showed that the bones xenografted with the Nog-KD clones have structural modifications indicative of bone formation/repair activity but not parental PC-3 cells and mock clones. Bone histomorphometry and pQCT further corroborated these findings. CONCLUSIONS Noggin expression by prostate cancer cells contribute to the induction of an osteolytic lesion. Conversely, shRNA-mediated suppression of noggin expression in the osteolytic cell line PC-3 restored bone formation and may represent a new therapeutic option for the treatment of osteolytic bone metastases. Bern, Switzerland© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e43 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chiara Secondini More articles by this author Antoinette Wetterwald More articles by this author Ruth Schwaninger More articles by this author Marco G. Cecchini More articles by this author George N. Thalmann More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Regulation of Oligodendrocyte Progenitor Cell Maturation by PPARδ: Effects on Bone Morphogenetic Proteins

In EAE (experimental autoimmune encephalomyelitis), agonists of PPARs (peroxisome proliferator-activated receptors) provide clinical benefit and reduce damage. In contrast with PPARγ, agonists of PPARδ are more effective when given at later stages of EAE and increase myelin gene expression, suggesting effects on OL (oligodendrocyte) maturation. In the present study we examined effects of the PPARδ agonist GW0742 on OPCs (OL progenitor cells), and tested whether the effects involve modulation of BMPs (bone morphogenetic proteins). We show that effects of GW0742 are mediated through PPARδ since no amelioration of EAE clinical scores was observed in PPARδ-null mice. In OPCs derived from E13 mice (where E is embryonic day), GW0742, but not the PPARγ agonist pioglitazone, increased the number of myelin-producing OLs. This was due to activation of PPARδ since process formation was reduced in PPARδ-null compared with wild-type OPCs. In both OPCs and enriched astrocyte cultures, GW0742 increased noggin protein expression; however, noggin mRNA was only increased in astrocytes. In contrast, GW0742 reduced BMP2 and BMP4 mRNA levels in OPCs, with lesser effects in astrocytes. These findings demonstrate that PPARδ plays a role in OPC maturation, mediated, in part, by regulation of BMP and BMP antagonists.

RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 microg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.

Noggin, impact on in vitro cell growth, expression pattern and the prognostic value in breast cancer.

Abstract Abstract #2084 Background: Noggin, a BMP (bone morphogenetic proteins) antagonist, has been demonstrated to play critical roles in regulation of biological functions of BMPs, members of the TGF-β superfamily and known regulators of bone metastasis in solid tumours including breast cancer. We have recently reported that the presence of endogenous noggin in cancer cells is a pivotal factor that governs the cells response to stimulation by bone morphogenetic protein, for example BMP-7. However, the role of noggin in breast cancer remains unclear. We presently investigated the role of Noggin in breast cancer cells and the pattern of expression in clinical breast tumours.
 Methods: The expression of Noggin mRNA and presence of Noggin protein was examined in a cohort of human breast cancer specimens (normal, n=33; cancer, n=112), using both quantitative real time PCR and immunohitochemical staining, respectively. The full-length human Noggin was cloned into a mammalian expression plasmid vector (pEF/His TOPO TA plasmid vector). The constructed Noggin expressing constructs were then transfected into breast cancer cells (MDA-MB-231). The expression of Noggin in the transfected cells was determined using RT-PCR and western blot. The effect on growth of MDA-MB-231 cells by Noggin was investigated using an in vitro growth assay.
 Results: Immunohistochemical staining revealed that Noggin protein was abundant in the cytoplasmic region of normal epithelial cells. Reduced staining of Noggin in breast cancer cells was seen when compared to normal breast tissue. Quantitative real time PCR further demonstrated that the decreasing Noggin expression correlated to poor prognosis, in that patients who remained disease-free showed a higher levels of the transcript (11113±3612) compared with those who had a poor clinical outcome (with metastasis and mortality, 6420±2241). Kaplan-Meier survival analysis showed that patients with high Noggin level had a longer survival (134.4 (121.3-147.5 - 95%CI) months) that those with low levels (102.8 (80.8-124.8) months). The absorbance of MDA-MB-231 Noggin over-expressing (MDA-MB-231Nogginexp) cells of Day 5 is 1.07±0.13, p<0.01 vs both MDA-MB-231 wild type (MDA-MB-231WT, 1.54±0.08) and empty vector control (MDA-MB-231pEF/His, 1.72±0.17).
 Conclusions: Noggin inhibits the growth of breast cancer cells and has a pattern of expression in that decreased Noggin expression correlates to poor prognosis of breast cancer patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2084.

FGF9-induced proliferative response to eosinophilic inflammation in oesophagitis

Background:Oesophagitis is characterised by basal cell hyperplasia and activated eosinophils, which release mediators including major basic protein (MBP). MBP and its mimetic polyarginine activate the calcium sensing receptor (CaSR) on...

Heparin Potentiates the in Vivo Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Bone morphogenetic protein 4: Potential regulator of shear stress-induced graft neointimal atrophy

Placement in baboons of a distal femoral arteriovenous fistula increases shear stress through aortoiliac polytetrafluoroethylene (PTFE) grafts and induces regression of a preformed neointima. Atrophy of the neointima might be controlled by shear stress-induced genes, including the bone morphogenetic proteins (BMPs). We have investigated the expression and function of BMPs 2, 4, and 5 in the graft neointima and in cultured baboon smooth muscle cells (SMCs). Baboons received bilateral aortoiliac PTFE grafts and 8 weeks later, a unilateral femoral arteriovenous fistula. Quantitative polymerase chain reaction showed that high shear stress increased BMP2, 4, and 5 messenger RNA (mRNA) in graft intima between 1 and 7 days, while noggin (a BMP inhibitor) mRNA was decreased. BMP4 most potently (60% inhibition) inhibited platelet-derived growth factor-stimulated SMC proliferation compared with BMP2 and BMP5 (31% and 26%, respectively). BMP4 also increased SMC death by 190% +/- 10%. Noggin reversed the antiproliferative and proapoptotic effects of BMP4. Finally, Western blotting confirmed BMP4 protein upregulation by high shear stress at 4 days. BMP4 expression demonstrated by in situ hybridization was confined to endothelial cells. Increased BMPs (particularly BMP4) coupled with decreased noggin may promote high shear stress-mediated graft neointimal atrophy by inhibiting SMC proliferation and increasing SMC death.