Biology of RANK, RANKL, and osteoprotegerin

The discovery of the receptor activator of nuclear factor-κB ligand (RANKL)/RANK/osteoprotegerin (OPG) system and its role in the regulation of bone resorption exemplifies how both serendipity and a logic-based approach can identify factors that regulate cell function. Before this discovery in the mid to late 1990s, it had long been recognized that osteoclast formation was regulated by factors expressed by osteoblast/stromal cells, but it had not been anticipated that members of the tumor necrosis factor superfamily of ligands and receptors would be involved or that the factors involved would have extensive functions beyond bone remodeling. RANKL/RANK signaling regulates the formation of multinucleated osteoclasts from their precursors as well as their activation and survival in normal bone remodeling and in a variety of pathologic conditions. OPG protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK. Thus, RANKL/OPG ratio is an important determinant of bone mass and skeletal integrity. Genetic studies in mice indicate that RANKL/RANK signaling is also required for lymph node formation and mammary gland lactational hyperplasia, and that OPG also protects arteries from medial calcification. Thus, these tumor necrosis factor superfamily members have important functions outside bone. Although our understanding of the mechanisms whereby they regulate osteoclast formation has advanced rapidly during the past 10 years, many questions remain about their roles in health and disease. Here we review our current understanding of the role of the RANKL/RANK/OPG system in bone and other tissues

Linking land restitution and urban development : lessons for restructuring the apartheid city from the Kipi land claim, Durban Metropolitan area.

Thesis (M.Dev.Studies)-University of Natal, Durban, 2003.This paper undertakes an indepth study of the Kipi land restitution claim. This study focuses on the nature of the settlement achieved in the case ofthe Kipi land claim and covers the period 1993 to 1999. It compares the Cato Manor reconstruction and development process and the Kipi land restoration and housing process within the Durban Metropolitan's Inner West Council area. The study does this by tracing the history of the Kipi community's relationship with the land, documenting the communities resistance of the removal in terms of the Group Areas Act and presenting a critical examination of the communities efforts to reclaim and develop their land. The study uses the case ~tudy method to analyse the principles embedded in this settlement and attempts to draw on these to inform possible policy recommendations in respect of other urban land claims. The central thesis of this dissertation argues that the quality of restitution delivery is directly affected by the degree to which it is located within local development coordination and management institutions and structures. In the Kipi claim the Council chose negotiation rather than the apartheid planning principles of prescription and coercion. This resulted in a integration of the housing and restitution processes. It is in this light that the role of the land claims working group which was set up by the Commission and the Durban Metro Inner West local council is evaluated. While in the Cato Manor case the Council chose to follow the legal route and opposed restoration in terms of section 34 of the Restitution ofLand Rights Act. The consequences of following the legal route has been that the housing and land restitution processes have been compartmentalised. It is argued that post apartheid planning is indeed a complex process that needs to engage creatively and flexibly with issues of over due social justice and the current development needs of the urban poor. It is important that in reconstructing the urban landscape that communities are involved in planning models that focus on bottom up processes for successful outcomes

Engineering Novel High-Resolution Bioelectronic Interfaces From Mxene Nanomaterials

At the interface between Man and Machine are electrode technologies. Using recording electrodes, it is possible to observe and monitor the activity of neurons or nervous tissue, affording us with an understanding of the basic dynamics underlying behavior and disease. By interacting with the nervous system through stimulating electrodes, it is possible to impact brain function, or evoke muscle activation and coordination, paving the way for treatments to severe neurological and neuromuscular disorders. However, despite the exciting promises of electrode technologies, current state-of-the-art platforms feature stiff and high-impedance materials, which are incompatible with soft biological tissue. Additionally, many current technologies suffer from shorter lifetimes than may be desirable for a truly chronic implant or wearable health monitoring device. Recently, there has been a shift in focus towards two-dimensional nanocarbons as alternative materials for superior electrode technologies. This comes as a result of the enhanced flexibility, biocompatibility, and electronic and electrochemical properties that most carbon-based nanomaterials exhibit. In particular, the 2D nanomaterial titanium carbide MXene (Ti3C2Tx) has very recently shown great promise for a variety of biomedical applications. However, the long-term stability of Ti3C2Tx has not been fully explored, and it is still unknown whether Ti3C2Tx can be used for chronic bioelectronic applications. Accordingly, in this thesis, I address and explore the key advantages of Ti3C2Tx for biopotential sensing, with a particular emphasis on validating this unique material for chronic recording studies. First, I demonstrate the superior advantages of Ti3C2Tx for direct recording of biopotential signals at the skin level in humans. Second, I define the long-term stability of Ti3C2Tx MXene in dried film form, and explore modifications in synthesis and film assembly to improve the material’s lifetime. Third, I fabricate and validate Ti3C2Tx-based epidermal sensors that exhibit comparable recording capabilities to state-of-the-art clinical electrodes, firmly establishing Ti3C2Tx electrode technologies for future, chronic experiments. The processing and fabrication methods developed herein translate into mature technologies with unique properties that are comparable to state-of-the-art designs, thereby offering a novel bioelectronic platform with the potential to benefit a variety of fields in both the research and clinical settings

A p21-Activated Kinase Is Required for Conidial Germination in Penicillium marneffei

Asexual spores (conidia) are the infectious propagules of many pathogenic fungi, and the capacity to sense the host environment and trigger conidial germination is a key pathogenicity determinant. Germination of conidia requires the de novo establishment of a polarised growth axis and consequent germ tube extension. The molecular mechanisms that control polarisation during germination are poorly understood. In the dimorphic human pathogenic fungus Penicillium marneffei, conidia germinate to produce one of two cell types that have very different fates in response to an environmental cue. At 25 °C, conidia germinate to produce the saprophytic cell type, septate, multinucleate hyphae that have the capacity to undergo asexual development. At 37 °C, conidia germinate to produce the pathogenic cell type, arthroconidiating hyphae that liberate uninucleate yeast cells. This study shows that the p21-activated kinase pakA is an essential component of the polarity establishment machinery during conidial germination and polarised growth of yeast cells at 37 °C but is not required for germination or polarised growth at 25 °C. Analysis shows that the heterotrimeric G protein α subunit GasC and the CDC42 orthologue CflA lie upstream of PakA for germination at both temperatures, while the Ras orthologue RasA only functions at 25 °C. These findings suggest that although some proteins that regulate the establishment of polarised growth in budding yeast are conserved in filamentous fungi, the circuitry and downstream effectors are differentially regulated to give rise to distinct cell types

Bone Remodeling and the Role of TRAF3 in Osteoclastic Bone Resorption

Skeletal health is maintained by bone remodeling, a process in which microscopic sites of effete or damaged bone are degraded on bone surfaces by osteoclasts and subsequently replaced by new bone, which is laid down by osteoblasts. This normal process can be disturbed in a variety of pathologic processes, including localized or generalized inflammation, metabolic and endocrine disorders, primary and metastatic cancers, and during aging as a result of low-grade chronic inflammation. Osteoclast formation and activity are promoted by factors, including cytokines, hormones, growth factors, and free radicals, and require expression of macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) by accessory cells in the bone marrow, including osteoblastic and immune cells. Expression of TNF receptor-associated factor 6 (TRAF6) is required in osteoclast precursors to mediate RANKL-induced activation of NF-κB, which is also necessary for osteoclast formation and activity. TRAF3, in contrast is not required for osteoclast formation, but it limits RANKL-induced osteoclast formation by promoting proteasomal degradation of NF-κB-inducing kinase in a complex with TRAF2 and cellular inhibitor of apoptosis proteins (cIAP). TRAF3 also limits osteoclast formation induced by TNF, which mediates inflammation and joint destruction in inflammatory diseases, including rheumatoid arthritis. Chloroquine and hydroxychloroquine, anti-inflammatory drugs used to treat rheumatoid arthritis, prevent TRAF3 degradation in osteoclast precursors and inhibit osteoclast formation in vitro. Chloroquine also inhibits bone destruction induced by ovariectomy and parathyroid hormone in mice in vivo. Mice genetically engineered to have TRAF3 deleted in osteoclast precursors and macrophages develop early onset osteoporosis, inflammation in multiple tissues, infections, and tumors, indicating that TRAF3 suppresses inflammation and tumors in myeloid cells. Mice with TRAF3 conditionally deleted in mesenchymal cells also develop early onset osteoporosis due to a combination of increased osteoclast formation and reduced osteoblast formation. TRAF3 protein levels decrease in bone and bone marrow during aging in mice and humans. Development of drugs to prevent TRAF3 degradation in immune and bone cells could be a novel therapeutic approach to prevent or reduce bone loss and the incidence of several common diseases associated with aging

Increased lymphangiogenesis in joints of mice with inflammatory arthritis

Angiogenesis is involved in the pathogenesis of inflammatory arthritis, but little is known about the role of lymphangiogenesis in this setting. Here, we examined whether tumor necrosis factor (TNF) stimulates osteoclast precursors (OCPs) to produce the lymphatic growth factor, vascular endothelial growth factor-C (VEGF-C), and induce lymphangiogenesis. We used TNF-transgenic (Tg) mice and mice with serum-induced arthritis. OCPs were purified by fluorescence-activated cell sorting of CD11b+/Gr-1-/lo blood or bone marrow cells and subjected to microarray analysis or were generated from spleen or joint cells and treated with TNF. Expression of VEGFs was analyzed and examined by real-time reverse transcription-polymerase chain reaction and Western blotting. Immunostaining and magnetic resonance imaging were used to quantify lymphatic vessels and volumes of synovium and draining lymph nodes. TNF stimulated VEGF-C expression by OCPs and increased nuclear factor-kappa B (NF-κB) binding to an NF-κB sequence in the VEGF-C promoter. OCPs from joints of TNF-Tg mice express high levels of VEGF-C. Lymphatic vessel numbers and size were markedly increased in joint sections of TNF-Tg mice and mice with serum-induced arthritis. The severity of synovitis correlated with draining lymph node size. In summary, TNF induces OCPs to produce VEGF-C through NF-κB, leading to significantly increased lymphangiogenesis in joints of arthritic mice. The lymphatic system may play an important role in the pathogenesis of inflammatory arthritis

Decreased C-Src Expression Enhances Osteoblast Differentiation and Bone Formation

c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src−/− relative to Src+/+ mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by ∼60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src−/− osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice

Lead Exposure Inhibits Fracture Healing and Is Associated with Increased Chondrogenesis, Delay in Cartilage Mineralization, and a Decrease in Osteoprogenitor Frequency

Lead exposure continues to be a significant public health problem. In addition to acute toxicity, Pb has an extremely long half-life in bone. Individuals with past exposure develop increased blood Pb levels during periods of high bone turnover or resorption. Pb is known to affect osteoblasts, osteoclasts, and chondrocytes and has been associated with osteoporosis. However, its effects on skeletal repair have not been studied. We exposed C57/B6 mice to various concentrations of Pb acetate in their drinking water to achieve environmentally relevant blood Pb levels, measured by atomic absorption. After exposure for 6 weeks, each mouse underwent closed tibia fracture. Radiographs were followed and histologic analysis was performed at 7, 14, and 21 days. In mice exposed to low Pb concentrations, fracture healing was characterized by a delay in bridging cartilage formation, decreased collagen type II and type X expression at 7 days, a 5-fold increase in cartilage formation at day 14 associated with delayed maturation and calcification, and a persistence of cartilage at day 21. Fibrous nonunions at 21 days were prevalent in mice receiving very high Pb exposures. Pb significantly inhibited ex vivo bone nodule formation but had no effect on osteoclasts isolated from Pb-exposed animals. No significant effects on osteoclast number or activity were observed. We conclude that Pb delays fracture healing at environmentally relevant doses and induces fibrous nonunions at higher doses by inhibiting the progression of endochondral ossification

CRTAP Is Required for Prolyl 3- Hydroxylation and Mutations Cause Recessive Osteogenesis Imperfecta

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