Osteocytes and mechanical loading: The Wnt connection

Bone adapts to the mechanical forces that it experiences. Orthodontic tooth movement harnesses the cell‐ and tissue‐level properties of mechanotransduction to achieve alignment and reorganization of the dentition. However, the mechanisms of action that permit bone resorption and formation in response to loads placed on the teeth are incompletely elucidated, though several mechanisms have been identified. Wnt/Lrp5 signalling in osteocytes is a key pathway that modulates bone tissue's response to load. Numerous mouse models that harbour knock‐in, knockout and transgenic/overexpression alleles targeting genes related to Wnt signalling point to the necessity of Wnt/Lrp5, and its localization to osteocytes, for proper mechanotransduction in bone. Alveolar bone is rich in osteocytes and is a highly mechanoresponsive tissue in which components of the canonical Wnt signalling cascade have been identified. As Wnt‐based agents become clinically available in the next several years, the major challenge that lies ahead will be to gain a more complete understanding of Wnt biology in alveolar bone so that improved/expedited tooth movement becomes a possibility

The Political Foundations of Ibn Bajjah's "Governance of the Solitary"

A first impression might lead one to characterize the "Governance of the Solitary," the most famous treatise by the medieval Arabic-Islamic philosopher Ibn Bājjah, as favoring radical individualism, and thus as breaking with the political orientation of ancient philosophy. In fact, the treatise returns to the wisdom of Plato and Aristotle, reaffirming the ancient principle that the human being is by nature political and that the highest life for the city and the individual is the same, the life of virtue pursued for the sake of happiness. For Ibn Bājjah, the highest goal intended for human beings by nature is political, namely, the perfect virtuous city. In the absence of the city oriented toward perfect virtue, the philosopher may find it necessary to lead a life of isolation. According to Ibn Bājjah, this solitary life seeks to preserve on behalf of the city the possibility of its deliverance from imperfection by pursuing the highest goal of the individual human being, namely, the attainment of conjunction with the divine intellect. By means of this intermediary goal, the solitary aims to deliver knowledge of perfect virtue to the city that is needed to bring about political happiness. Ibn Bājjah's account of the solitary shows that the philosopher does not abandon the city by pursuing philosophy in isolation, but in fact the isolated philosopher embodies the hope of bringing about the city's perfection. Practically speaking, this dissertation seeks to establish the second and third parts of the "Governance" as elaborations on the political teaching begun in the first part. To remain true to the author's intent, I argue that one cannot bypass Ibn Bājjah's concern for the perfect virtuous city in Part I, in order to present his teaching on governance as culminating in the life of the solitary described in Part III. The following study aims to take into account the treatise as a whole and to discuss it as faithful as possible to the original Arabic text

Mechanical Regulation of Wnt/β-catenin Signaling in Bone Cells

poster abstractThe Wnt/β-catenin signaling pathway is an important regulatory pathway in development and maintenance of various tissues, including bone. Active Wnt interacts with the frizzled/LRP receptor activating dishevelled, which in turn inactivates the GSK-3β complex and allows βcatenin to accumulate in the cytoplasm. β-catenin translocates to the nucleus where it activates a wide number of developmental target genes. Wnt can be sequestered by soluble frizzled related protein causing the inactivation of dishevelled, allowing for activation of the GSK-3β complex. This activated complex binds β-catenin and targets it for degradation. In addition to its other major role as a linker between cadherins and the actin cytoskeleton, β-catenin accumulation in the cytoplasm and subsequent translocation to the nucleus is a key step in the wnt/β-catenin signaling pathway. In bone, wnt/β-catenin signaling regulates skeletal formation, limb development and osteoblast maturation. Both active and inactive wnt/β-catenin signaling regulate bone cell development, active wnt/β-catenin signaling promotes osteoblast formation, while inactive wnt/β-catenin signaling inhibits osteoclast differentiation. Mechanical regulation of bone cells occurs through a process known as mechanotransduction which can be induced by fluid shear stress that occurs across the surfaces of osteoblasts and osteocytes, the effector cells of mechanotransduction. We hypothesize that knocking down β-catenin expression in mouse osteoblasts and osteoprogenitors will change the way these cells respond to fluid shear stress and regulate expression of relevant bone target genes. The future aims of this project are to assess the role of β-catenin during fluid shear stress induced osteoprogenitor cell differentiation by examining the expression of important osteoblast differentiation markers including: runx2, COX2, osteopontin, and osteocalcin and evaluate the significance of β-catenin during differentiation of bone marrow stromal cells

Work-life imbalance: informal care and paid employment

In the United Kingdom informal carers are people who look after relatives or friends who need extra support because of age, physical or learning disability or illness. The majority of informal carers are women and female carers also care for longer hours and for longer durations than men. Thus women and older women in particular, shoulder the burden of informal care. We consider the costs of caring in terms of the impact that these kinds of caring responsibilities have on employment. The research is based on the responses of informal carers to a dedicated questionnaire and in-depth interviews with a smaller sub-sample of carers. Our results indicate that the duration of a caring episode as well as the hours carers commit to caring impact on their employment participation. In addition carers’ employment is affected by financial considerations, the needs of the person they care for, carers’ beliefs about the compatibility of informal care and paid work and employers’ willingness to accommodate carers’ needs. Overall, the research confirms that informal carers continue to face difficulties when they try to combine employment and care in spite of recent policy initiatives designed to help them

Estrogen receptor-β regulates mechanical signaling in primary osteoblasts

Mechanical loading is an important regulator in skeletal growth, maintenance, and aging. Estrogen receptors have a regulatory role in mechanically induced bone adaptation. Estrogen receptor-α (ERα) is known to enhance load-induced bone formation, whereas ERβ negatively regulates this process. We hypothesized that ERβ regulates mechanical signaling in osteoblasts. We tested this hypothesis by subjecting primary calvarial cells isolated from wild-type and ERβ-knockout mice (BERKO) to oscillatory fluid flow in the absence or presence of estradiol (E2). We found that the known responses to fluid shear stress, i.e., phosphorylation of the mitogen-activated protein kinase ERK and upregulation of COX-2 expression, were inhibited in BERKO cells in the absence of E2. Flow-induced increase in prostaglandin E2 (PGE2) release was not altered in BERKO cells in the absence of E2, but was increased when E2 was present. Additionally, immunofluorescence analysis and estrogen response element luciferase assays revealed increased ERα expression and flow- and ligand-induced nuclear translocation as well as transcriptional activity in BERKO cells in both the presence and absence of E2. Taken together, these data suggest that ERβ plays both ligand-dependent and ligand-independent roles in mechanical signaling in osteoblasts. Furthermore, our data suggest that one mechanism by which ERβ regulates mechanotransduction in osteoblasts may result from its inhibitory effect on ERα expression and function. Targeting estrogen receptors (e.g., inhibiting ERβ) may represent an effective approach for prevention and treatment of age-related bone loss

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail

L-selectin mediated leukocyte tethering in shear flow is controlled by multiple contacts and cytoskeletal anchorage facilitating fast rebinding events

L-selectin mediated tethers result in leukocyte rolling only above a threshold in shear. Here we present biophysical modeling based on recently published data from flow chamber experiments (Dwir et al., J. Cell Biol. 163: 649-659, 2003) which supports the interpretation that L-selectin mediated tethers below the shear threshold correspond to single L-selectin carbohydrate bonds dissociating on the time scale of milliseconds, whereas L-selectin mediated tethers above the shear threshold are stabilized by multiple bonds and fast rebinding of broken bonds, resulting in tether lifetimes on the timescale of $10^{-1}$ seconds. Our calculations for cluster dissociation suggest that the single molecule rebinding rate is of the order of $10^4$ Hz. A similar estimate results if increased tether dissociation for tail-truncated L-selectin mutants above the shear threshold is modeled as diffusive escape of single receptors from the rebinding region due to increased mobility. Using computer simulations, we show that our model yields first order dissociation kinetics and exponential dependence of tether dissociation rates on shear stress. Our results suggest that multiple contacts, cytoskeletal anchorage of L-selectin and local rebinding of ligand play important roles in L-selectin tether stabilization and progression of tethers into persistent rolling on endothelial surfaces.Comment: 9 pages, Revtex, 4 Postscript figures include

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1- integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha- actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha- actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via α-actinin: Receptor positioning in microvilli does not require interaction with α-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein α-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified α-actinin to L-selectin (K(d) = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of α-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of α-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including α-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that α-actinin binds directly to L-selectin and that vinculin associates by binding to α-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with α-actinin or vinculin. Surprisingly, this mutant L- selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L- selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of α-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton

Lrp4 Mediates Bone Homeostasis and Mechanotransduction through Interaction with Sclerostin In Vivo

Wnt signaling plays a key role in regulating bone remodeling. In vitro studies suggest that sclerostin's inhibitory action on Lrp5 is facilitated by the membrane-associated receptor Lrp4. We generated an Lrp4 R1170W knockin mouse model (Lrp4KI), based on a published mutation in patients with high bone mass (HBM). Lrp4KI mice have an HBM phenotype (assessed radiographically), including increased bone strength and formation. Overexpression of a Sost transgene had osteopenic effects in Lrp4-WT but not Lrp4KI mice. Conversely, sclerostin inhibition had blunted osteoanabolic effects in Lrp4KI mice. In a disuse-induced bone wasting model, Lrp4KI mice exhibit significantly less bone loss than wild-type (WT) mice. In summary, mice harboring the Lrp4-R1170W missense mutation recapitulate the human HBM phenotype, are less sensitive to altered sclerostin levels, and are protected from disuse-induced bone loss. Lrp4 is an attractive target for pharmacological targeting aimed at increasing bone mass and preventing bone loss due to disuse