Remodeling Cycle Research Articles

Nuclear Export of Single Native mRNA

Export of nuclear mRNA is a key transport process in eukaryotic cells, but its dynamics and regulation is not well understood. We labelled native mRNA particles in living Chironomus tentans salivary gland cells [1, 2] using fluorescent hrp36, the C. tentans homolog of mammalian hnRNP A1, and the nuclear envelope by fluorescent NTF2. Using light sheet microscopy we succeeded to image single mRNA particles more than 100 micrometer deep in the sample [3] and traced for the first time single native mRNA particles across the nuclear envelope. The particles often probed nuclear pore complexes at their nuclear face with an average duration of τ ∼ 60 ms, but the actual export process was about ten-fold longer (τ ≈ 0.54 s) containing a distinct rate-limiting step. Analysis of single fluorescent Dbp5 proteins, the RNA helicase essential for mRNA export, revealed that Dbp5 was exclusively encountered at the cytoplasmic face of the nuclear pore complex with a binding duration of τ ∼ 50 ms. Our results suggest that single mRNA particles required >10 cycles of remodelling by Dbp5 to accomplish export directionality. [1] Siebrasse, J.-P., R. Veith, A. Dobay, H. Leonhardt, B. Daneholt and U. Kubitscheck. 2008. Proc Natl Acad Sci USA 105:20291-6. [2] Veith, R., T. Sorkalla, E. Baumgart, J. Anzt, H. Häberlein, S. Tyagi, J. P. Siebrasse, and U. Kubitscheck. 2010. Biophys. J. 99: 2676-2685. [3] Ritter, J.G., J. P. Siebrasse, R. Veith, A. Veenendaal, and U. Kubitscheck. 2010. PLoS ONE 5, e11639.

Premature loss of bone remodeling compartment canopies is associated with deficient bone formation: A study of healthy individuals and patients with cushing's syndrome

A remarkable property of bone remodeling is that osteoblasts form bone matrix exactly where and when osteoclasts have removed it. The bone remodeling compartment (BRC) canopies that cover bone surfaces undergoing remodeling were proposed to be critical players in this mechanism. Here, we provide support to this hypothesis by analyzing the changes in prevalence of BRC canopies during the progress of the remodeling cycle in a cohort of healthy individuals and in patients with endogenous Cushing's syndrome (CS), and by relating these changes in prevalence with the extent of bone forming surfaces. Both cohorts showed almost 100% canopy coverage above resorbing osteoclasts, and only about 76% above bone forming surfaces. This indicates that BRC canopies are invariably associated with the early stage of the remodeling cycle, but may disappear later. Interestingly, in control and two-thirds of the CS patients, a significant decline in canopy coverage occurred only once bone formation was initiated, but in the remaining third of the CS patients the prevalence of canopies already decreased before bone formation. This canopy loss before initiation of bone formation coincided with significantly less bone-forming surface compared with canopy loss at a later stage. These observations support a model where bone restitution is compromised in the absence of BRC canopies, and apparently does not start when the BRC canopy is lost before initiation of the bone formation step. This model is discussed in the context of possible biological roles of BRC canopies. It suggests that BRC canopies could be privileged targets for treating patients suffering from a negative bone formation-resorption balance.

Efficient Construction of Homozygous Diploid Strains Identifies Genes Required for the Hyper-Filamentous Phenotype in Saccharomyces cerevisiae

Yeast cells undergo diploid-specific developments such as spore formation via meiosis and pseudohyphal development under certain nutrient-limited conditions. Studies on these aspects require homozygous diploid mutants, which are generally constructed by crossing strains of opposite mating-type with the same genetic mutation. So far, there has been no direct way to generate and select diploids from haploid cells. Here, we developed a method for efficient construction of homozygous diploids using a PGAL1-HO gene (galactose-inducible mating-type switch) and a PSTE18-URA3 gene (counter selection marker for diploids). Diploids are generated by transient induction of the HO endonuclease, which is followed by mating of part of the haploid population. Since the STE18 promoter is repressed in diploids, diploids carrying PSTE18-URA3 can be selected on 5-fluoroorotic acid (5-FOA) plates where the uracil prototrophic haploids cannot grow. To demonstrate that this method is useful for genetic studies, we screened suppressor mutations of the complex colony morphology, strong agar invasion and/or hyper-filamentous growth caused by lack of the Hog1 MAPK in the diploid Σ1278b strain background. Following this approach, we identified 49 suppressor mutations. Those include well-known positive regulator genes for filamentous growth signaling pathways, genes involved in mitochondrial function, DNA damage checkpoint, chromatin remodeling, and cell cycle, and also previously uncharacterized genes. Our results indicate that combinatorial use of the PGAL1-HO and PSTE18-URA3 genes is suitable to efficiently construct and select diploids and that this approach is useful for genetic studies especially when combined with large-scale screening.

Working Without Kinase Activity: Phosphotransfer-Independent Functions of Extracellular Signal–Regulated Kinases

The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) and ERK2 play well-characterized roles in the regulation of key cellular processes, such as proliferation, differentiation, and survival, by acting as serine and threonine kinases in the phosphorylation of ~200 substrates that are distributed in different subcellular localizations. However, over the past few years, evidence has mounted that indicates that the mechanism of action of ERK1 and ERK2 may extend beyond their role as canonical kinases. For example, proteins such as poly(ADP-ribose) polymerase 1, topoisomerase II, and MAPK phosphatase 3 (MKP-3) are activated by a direct interaction with ERK2 that does not involve any phosphotransfer activity. In addition, ERK2 binds to DNA and acts as a transcriptional regulator independently of its function as a kinase. Moreover, other studies demonstrate that ERK1 and ERK2 can regulate cell cycle entry by disrupting the interaction between the retinoblastoma pocket protein and lamin A in a kinase-independent fashion. These findings strongly support the notion that ERK1 and ERK2 can play functionally important roles independently of their regular catalytic activities and provide the basis for a new perspective from which to view these hitherto archetypical signaling kinases.

Fabrication of Carbonate Apatite Block from Calcium Sulfate by Hydrothermal Treatment

Carbonate apatite (CO3Ap) is expected to be an ideal bone substitute since it can harmonize with the bone remodeling cycle. The aim of this study is to fabricate a CO3Ap bone substitute from gypsum (calcium sulfate, CaSO4·2H2O) hardening bodies based on dissolution-precipitation reaction. Calcium sulfate hemihydrate mixed with water at a water-to-powder ratio of 0.5 was packed in a split stainless mold and kept at room temperature for 24 hours to obtain set CaSO4·2H2O. The set CaSO4·2H2O was hydrothermally treated in the presence of disodium hydrogen phosphate (Na2HPO4) and sodium hydrogen carbonate (NaHCO3). The results of powder X-ray diffraction and Fourier transform infrared spectroscopy indicated that CO3Ap block could be fabricated from the set CaSO4·2H2O block by hydrothermal treatment with Na2HPO4 and NaHCO3. When the treatment temperature was increased, the conversion rate to CO3Ap increased. However, the carbonate content decreased with increasing treatment temperature.

Biomimetic Matrices Self-Initiating the Induction of Bone Formation

The new strategy of tissue engineering, and regenerative medicine at large, is to construct biomimetic matrices to mimic nature's hierarchical structural assemblages and mechanisms of simplicity and elegance that are conserved throughout genera and species. There is a direct spatial and temporal relationship of morphologic and molecular events that emphasize the biomimetism of the remodeling cycles of the osteonic corticocancellous bone versus the "geometric induction of bone formation," that is, the induction of bone by "smart" concavities assembled in biomimetic matrices of macroporous calcium phosphate-based constructs. The basic multicellular unit of the corticocancellous bone excavates a trench across the bone surface, leaving in its wake a hemiosteon rather than an osteon, that is, a trench with cross-sectional geometric cues of concavities after cyclic episodes of osteoclastogenesis, eventually leading to osteogenesis. The concavities per se are geometric regulators of growth-inducing angiogenesis and osteogenesis as in the remodeling processes of the corticocancellous bone. The concavities act as a powerful geometric attractant for myoblastic/myoendothelial and/or endothelial/pericytic stem cells, which differentiate into bone-forming cells. The lacunae, pits, and concavities cut by osteoclastogenesis within the biomimetic matrices are the driving morphogenetic cues that induce bone formation in a continuum of sequential phases of resorption/dissolution and formation. To induce the cascade of bone differentiation, the soluble osteogenic molecular signals of the transforming growth factor β supergene family must be reconstituted with an insoluble signal or substratum that triggers the bone differentiation cascade. By carving a series of repetitive concavities into solid and/or macroporous biomimetic matrices of highly crystalline hydroxyapatite or biphasic hydroxyapatite/β-tricalcium phosphate, we were able to embed smart biologic functions within intelligent scaffolds for tissue engineering of bone. The concavities assembled in the bioceramic constructs biomimetize the remodeling cycle of the corticocancellous bone and are endowed with multifunctional pleiotropic self-assembly capacities, initiating angiogenesis and bone formation by induction without the exogenous applications of the osteogenic-soluble molecular signals of the transforming growth factor β supergene family. The incorporation of specific biologic activities into biomimetic matrices by manipulating the geometry of the substratum, defined as geometric induction of bone formation, is now helping to engineer therapeutic osteogenesis in clinical contexts.

A model for the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw and teriparatide's potential role in its resolution

The objective of this study was to present a comprehensive model for the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw (BON). Review of PubMed literature relevant to BON, bisphosphonates (BPs), and bone remodeling. Six case reports of spontaneous resolution of BON lesions following administration of teriparatide (Forteo; Eli Lilly and Co., Indianapolis, IN) were identified. These reports suggest that osteoanabolic therapies may hold promise in BON management. Here we propose that BON pathogenesis is multifactorial and is the combined result of attenuated osteoblastic activity (owing to the patient's underlying disease, e.g., osteoporosis or multiple myeloma), BP-mediated osteoclast toxicity, and the resultant compromised osteoblast-osteoclast interactions during bone remodeling. Consequently, a vicious cycle of ineffective local remodeling results in the persistence of defective bone, compromised tissue perfusion, and if unresolved, ultimately leads to necrosis. Our model for BON pathogenesis advocates for earlier therapeutic intervention in BON. The biological rationale for teriparatide's efficacy in BON justifies further investigation.

Overview of Osteoarthritis

Osteoarthritis is the most common type of arthritis thoughout the world. It is among the leading causes of disability and functional loss. Given the aging of the population and the rise in obesity which is a major risk factor for knee and hip osteoarthritis, the prevalence of osteoarthritis is increasing. Painful osteoarthritis of the knee affects at least 12% of persons age 60 and over in the Western World and likely affects an even higher percentage of persons this age in China. While hyaline articular cartilage loss is the signature feature on pathology, the disease affects all structures of the joint in a nonuniform manner, affecting some areas of the joint more than others. Bone remodeling, fibrocartilage (especially meniscal) degeneration, synovitis, and weakness of muscles that bridge the joint all occur at the same time as cartilage loss. While disease begins perhaps with focal cartilage erosion or injury, the joint narrows at the site of erosion, and stress across this region of the joint increases, causing more damage. Misalignment across the joint creates high local stresses which are particularly damaging, pointing to the critical role of mechanical loading in driving the disease process. A vicious cycle of progressive local cartilage loss, bone remodeling, meniscal change, synovitis and misalignment across the joint emerges. Early on in this pathologic sequence, the person has little if any joint pain. Intermittent and mild joint pain starts usually only after considerable joint damage is present. Even after pathology is present, many persons have no joint pain and others develop pain with little evidence of pathology on x-ray. The structures in the joint whose pathology is tied to joint pain include bone and synovium and possibly tears in fibrocartilage or ligaments. Bone marrow lesions on MRI which histologically represent areas of bone damage are probably caused by malalignment related stress. These bone marrow lesions probably cause pain as does synovitis which is present in many osteoarthritic joints. Synovitis can produce fluid which distends the joint capsule which can also cause pain. The course of osteoarthritis is highly variable, but when progression occurs, it is often accompanied by more severe and more consistent joint pain. There are few, if any, effective preventive strategies for osteoarthritis. The main nonsurgical treatments for osteoarthritis are rehabilitative treatments including exercise, strengthening, weight loss, and braces and orthotics. Drug treatments include analgesics and antinflammatory medications. Nutriceuticals and hyaluronans are controversial in terms of efficacy. No treatment has been shown to slow the disease process.

Transcript profiling of crown rootless1 mutant stem base reveals new elements associated with crown root development in rice

BackgroundIn rice, the major part of the post-embryonic root system is made of stem-derived roots named crown roots (CR). Among the few characterized rice mutants affected in root development, crown rootless1 mutant is unable to initiate crown root primordia. CROWN ROOTLESS1 (CRL1) is induced by auxin and encodes an AS2/LOB-domain transcription factor that acts upstream of the gene regulatory network controlling CR development.ResultsTo identify genes involved in CR development, we compared global gene expression profile in stem bases of crl1 mutant and wild-type (WT) plants. Our analysis revealed that 250 and 236 genes are down- and up-regulated respectively in the crl1 mutant. Auxin induces CRL1 expression and consequently it is expected that auxin also alters the expression of genes that are early regulated by CRL1. To identify genes under the early control of CRL1, we monitored the expression kinetics of a selected subset of genes, mainly chosen among those exhibiting differential expression, in crl1 and WT following exogenous auxin treatment. This analysis revealed that most of these genes, mainly related to hormone, water and nutrient, development and homeostasis, were likely not regulated directly by CRL1. We hypothesized that the differential expression for these genes observed in the crl1 mutant is likely a consequence of the absence of CR formation. Otherwise, three CRL1-dependent auxin-responsive genes: FSM (FLATENNED SHOOT MERISTEM)/FAS1 (FASCIATA1), GTE4 (GENERAL TRANSCRIPTION FACTOR GROUP E4) and MAP (MICROTUBULE-ASSOCIATED PROTEIN) were identified. FSM/FAS1 and GTE4 are known in rice and Arabidopsis to be involved in the maintenance of root meristem through chromatin remodelling and cell cycle regulation respectively.ConclusionOur data showed that the differential regulation of most genes in crl1 versus WT may be an indirect consequence of CRL1 inactivation resulting from the absence of CR in the crl1 mutant. Nevertheless some genes, FAS1/FSM, GTE4 and MAP, require CRL1 to be induced by auxin suggesting that they are likely directly regulated by CRL1. These genes have a function related to polarized cell growth, cell cycle regulation or chromatin remodelling. This suggests that these genes are controlled by CRL1 and involved in CR initiation in rice.

P2-110 Number of teeth is associated with the level of salivary nitric oxide

IntroductionNitric oxide (NO) is involved in second messenger formation, osteoblast and osteoclast function, and blood flow. This raises the question of whether or not altered salivary NO levels interfere with...

Osteoporosis: A Paradox in Ankylosing Spondylitis

Ankylosing spondylitis is a chronic and severe inflammatory disease of the axial skeleton and the joints. Inflammation is associated with trabecular bone loss leading to osteoporosis but also with corcal new bone formation leading to progressive ankylosis of the spine and sacroiliac joints. This results in an apparent paradox of bone formation and loss taking place at sites closesly located to each other. Osteoporosis can be explained by the impact of inflammation of the bone remodeling cycle. In contrast, new bone formation has been linked to aberrant acvaon of bone morphogenec protein and Wnt signaling. In this commentary, we review recent data on this bone paradox and highlight recent advances including the effect of current drug therapies and the idenfication of new therapeutic targets.

The Dbp5 cycle at the nuclear pore complex during mRNA export II: nucleotide cycling and mRNP remodeling by Dbp5 are controlled by Nup159 and Gle1

Essential messenger RNA (mRNA) export factors execute critical steps to mediate directional transport through nuclear pore complexes (NPCs). At cytoplasmic NPC filaments, the ATPase activity of DEAD-box protein Dbp5 is activated by inositol hexakisphosphate (IP(6))-bound Gle1 to mediate remodeling of mRNA-protein (mRNP) complexes. Whether a single Dbp5 executes multiple remodeling events and how Dbp5 is recycled are unknown. Evidence suggests that Dbp5 binding to Nup159 is required for controlling interactions with Gle1 and the mRNP. Using in vitro reconstitution assays, we found here that Nup159 is specifically required for ADP release from Dbp5. Moreover, Gle1-IP(6) stimulates ATP binding, thus priming Dbp5 for RNA loading. In vivo, a dbp5-R256D/R259D mutant with reduced ADP binding bypasses the need for Nup159 interaction. However, NPC spatial control is important, as a dbp5-R256D/R259D nup42Δ double mutant is temperature-sensitive for mRNA export. Further analysis reveals that remodeling requires a conformational shift to the Dbp5-ADP form. ADP release factors for DEAD-box proteins have not been reported previously and reflect a new paradigm for regulation. We propose a model wherein Nup159 and Gle1-IP(6) regulate Dbp5 cycles by controlling its nucleotide-bound state, allowing multiple cycles of mRNP remodeling by a single Dbp5 at the NPC.

Bone metastasis in prostate cancer: emerging therapeutic strategies

Metastatic bone disease (MBD) in advanced-stage cancer increases the risk of intractable bone pain, pathological skeletal fracture, spinal-cord compression and decreased survival. The disease manifestation course during MBD is largely driven by homotypic and heterotypic cellular interactions between invading tumor cells, osteoblasts and osteoclasts. The outcome is a sustained vicious cycle of bone matrix remodeling. Osteoclast-mediated bone degradation and subsequent bone loss are the hallmarks of secondary bone metastases from most solid tumors. An additional complication in prostate cancer is the predominance of osteosclerotic lesions typified by inappropriate bone production. Successful therapeutic strategies for the treatment of osteolytic MBD include the administration of intravenous bisphosphonates or subcutaneous inhibitors of receptor activator of nuclear factor κB ligand (RANKL). Inhibitors of SRC and cABL kinases and cathepsin K are under clinical investigation as potential anti-osteolytics. In contrast to the rapid progress being made in the development of anti-osteolytic therapies, the treatment of osteosclerotic MBD remains restricted to palliative radiotherapy for symptomatic solitary lesions and systemic taxane-based chemotherapy for widespread multiple lesions. This Review discusses the complex pathology of bone lesions in metastatic castration-resistant prostate cancer and focuses on new therapeutic strategies and targets that are emerging in preclinical studies.

Bone remodelling at a glance

The bone remodelling cycle (see Poster panel “The bone remodelling cycle”) maintains the integrity of the skeleton through the balanced activities of its constituent cell types. These are the bone-forming osteoblast, a cell that produces the organic bone matrix and aids its mineralisation ([

Disorders of Bone Remodeling

The skeleton provides mechanical support for stature and locomotion, protects vital organs, and controls mineral homeostasis. A healthy skeleton must be maintained by constant bone modeling to carry out these crucial functions throughout life. Bone remodeling involves the removal of old or damaged bone by osteoclasts (bone resorption) and the subsequent replacement of new bone formed by osteoblasts (bone formation). Normal bone remodeling requires a tight coupling of bone resorption to bone formation to guarantee no alteration in bone mass or quality after each remodeling cycle. However, this important physiological process can be derailed by a variety of factors, including menopause-associated hormonal changes, age-related factors, changes in physical activity, drugs, and secondary diseases, which lead to the development of various bone disorders in both women and men. We review the major diseases of bone remodeling, emphasizing our current understanding of the underlying pathophysiological mechanisms.

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

Hormonal regulation of uterine chemokines and immune cells

The ultimate function of the endometrium is to allow the implantation of a blastocyst and to support pregnancy. Cycles of tissue remodeling ensure that the endometrium is in a receptive state during the putative 'implantation window', the few days of each menstrual cycle when an appropriately developed blastocyst may be available to implant in the uterus. A successful pregnancy requires strict temporal regulation of maternal immune function to accommodate a semi-allogeneic embryo. To preparing immunological tolerance at the onset of implantation, tight temporal regulations are required between the immune and endocrine networks. This review will discuss about the action of steroid hormones on the human endometrium and particularly their role in regulating the inflammatory processes associated with endometrial receptivity.

J025023 シグナル伝達機構を考慮した骨梁リモデリングシミュレーション([J02502]生命体統合シミュレーション(2))

ignaling molecules expressed by osteocytes, osteoclasts and osteoblasts play important roles in adaptive bone remodeling, however, the mechanism by which these signaling pathways result in the changes of trabecular morphology still remains unclear. In this study, we propose mathematical models for trabecular remodeling considering signaling pathways, and conduct computer simulations using finite element method. Focusing on the movement of remodeling packets on the trabecular surface, we discuss the influence of signaling molecules on the morphological changes of trabeculae based on the trabecular remodeling simulation. First, higher RANKL expression rate results in the higher turnover of the bone remodeling cycle. Second, movement of the remodeling packets, formed by coupling of osteoclasts and osteoblasts, toward unnecessary trabecular bone surface area was observed in which osteoclast precursors sense low OPG concentration.

New insights into cathepsin D in mammary tissue development and remodeling

Cathepsin D (CatD) is a lysosomal aspartyl endopeptidase originally considered a "house keeping enzyme" involved in the clearance of unwanted proteins. However, recent studies have revealed CatD's involvement in apoptosis and autophagy, thus signifying an important function in the proper development and maintenance of multi-cellular organs. In the mammary gland, matrix degradation and the remodeling process are orchestrated by proteolytic enzymes, but the role of CatD at distinct developmental stages has remained mostly unexplored. Based on our previous studies we sought to address the role of this endopeptidase in mammary gland development and remodeling. By employing a mouse model, we report a previously unidentified participation of CatD in different stages of mammary gland development. Our findings reveal that CatD undergoes distinct protein processing at different stages of mammary gland development, and this customized processing results in differential enzymatic activity (constitutive and low pH activatable) best fitting particular stage(s) of development. In addition, at the onset of involution the N-glycan structure of this endopeptidase switches from a mixed high mannose and hybrid structure to an almost exclusively high mannose type, but reverts back to the original N-glycan composition by day 4 of involution. Our findings illuminate (at least in part) the "raison d'être" for CatD's numerous and highly regulated proteolytic processing steps from the pro-form to the mature enzyme. In the mammary gland, specific cleavage product(s) perform specialized function(s) befitting each stage of remodeling. It is noteworthy that deregulated synthesis, secretion and glycosylation of CatD are hallmarks of cancer progression. Thus, identifying the role of CatD in a dynamic normal tissue undergoing highly regulated cycles of remodeling could provide valuable information illuminating the deregulation of CatD associated with cancer development and metastasis.

The cell wall integrity checkpoint: coordination between cell wall synthesis and the cell cycle

The cell wall is an essential cellular component for the survival of fungi. The cell wall of the budding yeast Saccharomyces cerevisiae undergoes remodelling during the cell cycle. Evidence is accumulating that there are regulatory mechanisms that link cell wall remodelling and cell cycle progression. Here, we review one such mechanism, known as the 'cell wall integrity checkpoint', which functions to control cell cycle progression in response to cell wall perturbation.