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Background and Objective: While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. Material and Methods: To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a b-galactosidase transgene under the control of a LEF/TCF and b-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the b-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and b-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. Results: In these in vitro models, we showed that amelogenin can activate b-catenin signaling. Conclusion: Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone. [ABSTRACT FROM AUTHOR]

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin-binding proteins, that the post-translational up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form, according to the 2D gel immunoblotting assay. The up-regulation of cofilin-1 was also detected in aged mammalian tissues. The over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin-1, which led to a decrease in phosphorylated cofilin-1. The senescence induced by the over-expression of cofilin-1 was dependent on p27 Kip1 , but not on the p53 and p16 INK4 expressions. The knockdown of p27 Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over-expression of cofilin-1 induced the expression of p27 Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27 Kip1  gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down-regulation of TEAD1 was accompanied by the up-regulation of cofilin-1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27 Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin-1/TEAD1/p27 Kip1 signaling is involved in senescence-related morphological change and growth arrest., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Structural analysis of microscopic objects is a longstanding topic in several scientific disciplines, such as biological, mechanical, and materials sciences. The scanning electron microscope (SEM), as a promising imaging equipment has been around for decades to determine the surface properties (e.g., compositions or geometries) of specimens by achieving increased magnification, contrast, and resolution greater than one nanometer. Whereas SEM micrographs still remain two-dimensional (2D), many research and educational questions truly require knowledge and facts about their three-dimensional (3D) structures. 3D surface reconstruction from SEM images leads to remarkable understanding of microscopic surfaces, allowing informative and qualitative visualization of the samples being investigated. In this contribution, we integrate several computational technologies including machine learning, contrario methodology, and epipolar geometry to design and develop a novel and efficient method called 3DSEM++ for multi-view 3D SEM surface reconstruction in an adaptive and intelligent fashion. The experiments which have been performed on real and synthetic data assert the approach is able to reach a significant precision to both SEM extrinsic calibration and its 3D surface modeling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The heavy metal lead (Pb) has a deleterious effect on skeletal health. Because bone mass is maintained through a balance of bone formation and resorption, it is important to understand the effect of Pb levels on osteoblastic and osteoclastic activity. Pb exposure is associated with low bone mass in animal models and human populations; however, the correlation between Pb dosing and corresponding bone mass has been poorly explored. Thus, mice were exposed to increasing Pb and at higher levels (500 ppm), there was unexpectedly an increase in femur-tibial bone mass by 3 months of age. This is contrary to several studies alluded to earlier. Increased bone volume (BV) was accompanied by a significant increase in cortical thickness of the femur and trabecular bone that extended beyond the epiphyseal area into the marrow cavity. Subsequent evaluations revealed an increase in osteoclast numbers with high Pb exposure, but a deficiency in osteoclastic activity. These findings were substantiated by observed increases in levels of the resorption-altering hormones calcitonin and estrogen. In addition we found that pro-osteoclastic nuclear factor-kappa beta (NF-κB) pathway activity was dose dependently elevated with Pb, both in vivo and in vitro. However, the ability of osteoclasts to resorb bone was depressed in the presence of Pb in media and within test bone wafers. These findings indicate that exposure to high Pb levels disrupts early life bone accrual that may involve a disruption of osteoclast activity. This study accentuates the dose dependent variation in Pb exposure and consequent effects on skeletal health., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples.

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/β-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the β-catenin pathway using a pharmacological inhibitor of GSK-3β ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFβ canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/β-catenin pathway., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Förster resonance energy transfer (FRET) is a nonradiative process for the transfer of energy from an optically excited donor molecule (D) to an acceptor molecule (A) in the ground state. The underlying theory predicting the dependence of the FRET efficiency on the sixth power of the distance between D and A has stood the test of time. In contrast, a comprehensive kinetic-based theory developed recently for FRET efficiencies among multiple donors and acceptors in multimeric arrays has waited for further testing. That theory has been tested in the work described in this article using linked fluorescent proteins located in the cytoplasm and at the plasma membrane of living cells. The cytoplasmic constructs were fused combinations of Cerulean as donor (D), Venus as acceptor (A), and a photo-insensitive molecule (Amber) as a nonfluorescent (N) place holder: namely, NDAN, NDNA, and ADNN duplexes, and the fully fluorescent quadruplex ADAA. The membrane-bound constructs were fused combinations of GFP2 as donor (D) and eYFP as acceptor (A): namely, two fluorescent duplexes (i.e., DA and AD) and a fluorescent triplex (ADA). According to the theory, the FRET efficiency of a multiplex such as ADAA or ADA can be predicted from that of analogs containing a single acceptor (e.g., NDAN, NDNA, and ADNN, or DA and AD, respectively). Relatively small but statistically significant differences were observed between the measured and predicted FRET efficiencies of the two multiplexes. While elucidation of the cause of this mismatch could be a worthy endeavor, the discrepancy does not appear to question the theoretical underpinnings of a large family of FRET-based methods for determining the stoichiometry and quaternary structure of complexes of macromolecules in living cells., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Despite extensive research in knee and hip osteoarthritis (OA), the underlying mechanism of temporomandibular joint (TMJ) disorder remains largely unknown. The purpose of this study was to determine whether the constitutive activation of β-catenin in the middle and deep layers of the articular cartilage can compromise the homeostasis of this tissue in the TMJ. Col2CreERT2 transgenic mice were bred with RosamT/mG reporter mice to determine Cre recombination efficiency. Col2CreERT2 mice were then crossed with β-cateninflox(ex3)+ mice to generate β-catenin conditional activation mice, β-catenin(ex3)Col2ER. TMJ samples were harvested when the mice were 1-, 3- or 6-month-old and evaluated using histology, histomorphometry and immunohistochemistry. β-catenin(ex3)Col2ER mice were further crossed with Mmp13flox/flox and Adamts5-/- mice to generate (β-catenin(ex3)/Mmp13)Col2ER and β-catenin(ex3)Col2ER)/Adamts5-/- double mutant mice to investigate the role of Mmp13 and Adamts5 in the development of TMJ disorder. High levels of Cre-recombination were seen in Col2CreERT2;RosamT/mGmice. Progressive TMJ defects developed in 1-, 3- and 6-month-old β-catenin(ex3)Col2ER mice, as revealed by histology and histomorphometry. Results further demonstrated that the defects observed in β-catenin(ex3)Col2ER mice were significantly decelerated after deletion of the Mmp13 or Adamts5 gene in (β-catenin(ex3)/Mmp13)Col2ER or β-catenin(ex3)Col2ER/Adamts5-/- double mutant mice. In summary, we found that β-catenin is a critical gene in the induction of TMJ cartilage degeneration, and over-expressing β-catenin in TMJ cartilage leads to defects assembling an OA-like phenotype. Deletion of Mmp13 and Adamts5 in β-catenin(ex3)Col2ER mice ameliorates the development of TMJ defects. This study suggests that Mmp13 and Adamts5 could be potential therapeutic targets for the treatment of TMJ disorders.

Multiphoton micro-spectroscopy, employing diffraction optics and electron-multiplying CCD (EMCCD) cameras, is a suitable method for determining protein complex stoichiometry, quaternary structure, and spatial distribution in living cells using Förster resonance energy transfer (FRET) imaging. The method provides highly resolved spectra of molecules or molecular complexes at each image pixel, and it does so on a timescale shorter than that of molecular diffusion, which scrambles the spectral information. Acquisition of an entire spectrally resolved image, however, is slower than that of broad-bandwidth microscopes because it takes longer times to collect the same number of photons at each emission wavelength as in a broad bandwidth. Here, we demonstrate an optical micro-spectroscopic scheme that employs a laser beam shaped into a line to excite in parallel multiple sample voxels. The method presents dramatically increased sensitivity and/or acquisition speed and, at the same time, has excellent spatial and spectral resolution, similar to point-scan configurations. When applied to FRET imaging using an oligomeric FRET construct expressed in living cells and consisting of a FRET acceptor linked to three donors, the technique based on line-shaped excitation provides higher accuracy compared to the point-scan approach, and it reduces artifacts caused by photobleaching and other undesired photophysical effects.

Parathyroid hormone (PTH) plays a critical role in the regulation of chondrogenesis. In this study, we have found for the first time that Runt-related transcription factor 1 (Runx1) contributes to PTH-induced chondrogenesis. Upon PTH treatment, limb bud mesenchymal progenitor cells in micromass culture showed an enhanced chondrogenesis, which was associated with a significant increase of chondrogenic marker gene expression, such as type II collagen and type X collagen. Runx1 was also exclusively expressed in cells treated with PTH at the onset stage of chondrogenesis. Knockdown of Runx1 completely blunted PTH-mediated chondrogenesis. Furthermore, PTH induced Runx1 expression and chondrogenesis were markedly reduced by inhibition of protein kinase A (PKA) signaling. Taken together, our present study indicates that chondrogenesis induced by PTH in mesenchymal progenitor cells is mediated by Runx1, which involves the activation of PKA. These data provide a novel insight into understanding the molecular mechanisms behind PTH-enhanced cartilage regeneration.

The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M(3)Rs (muscarinic M(3) acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M(3)R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.

Lead remains a significant environmental toxin, and we believe we may have identified a novel target of lead toxicity in articular chondrocytes. These cells are responsible for the maintenance of joint matrix, and do so under the regulation of TGF-β signaling. As lead is concentrated in articular cartilage, we hypothesize that it can disrupt normal chondrocyte phenotype through suppression of TGF-β signaling. These experiments examine the effects of lead exposure in vivo and in vitro at biologically relevant levels, from 1 nM to 10 µM on viability, collagen levels, matrix degrading enzyme activity, TGF-β signaling, and articular surface morphology. Our results indicate that viability was unchanged at levels ≤100 µM Pb, but low and high level lead in vivo exposure resulted in fibrillation and degeneration of the articular surface. Lead treatment also decreased levels of type II collagen and increased type X collagen, in vivo and in vitro. Additionally, MMP13 activity increased in a dose-dependent manner. Active caspase 3 and 8 were dose-dependently elevated, and treatment with 10 µM Pb resulted in increases of 30% and 500%, respectively. Increasing lead treatment resulted in a corresponding reduction in TGF-β reporter activity, with a 95% reduction at 10µM. Levels of phosphoSmad2 and 3 were suppressed in vitro and in vivo and lead dose-dependently increased Smurf2. These changes closely parallel those seen in osteoarthritis. Over time this phenotypic shift could compromise maintenance of the joint matrix., (Copyright © 2012 Orthopaedic Research Society.)

The key to treating steroid-induced necrosis of femoral heads (SINFH) is early diagnosis. Dramatic improvements in diagnosis could be made if the pathogenesis of SINFH was more fully understood; however, the underlying mechanism of this disease is currently unknown. To explore the potential mechanism of SINFH, we performed gene array analysis on a rat model of the disease and compare the expression profile with that of normal rats. A quantitative RT-PCR and immunohistochemistry (IHC) assays were used to confirm the microarray results. Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (deposited in GenBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and transcription related genes. The results of quantitative RT-PCR and IHC were consistent with gene chip results. Our findings indicate that many genes involved in diverse signaling pathways were differentially expressed between SINFH rats and normal rats. Furthermore, our findings suggest that the development of SINFH is a complicated and dynamic process affected by multiple factors and signaling pathways and regulated by various genes.

Background: Osteoporosis is a growing worldwide problem, with the greatest burden resulting from fractures. Nevertheless, the majority of fractures in adults occur in those with "osteopenia" (bone mineral density (BMD) only moderately lower than young normal individuals). Since long-term drug therapy is an expensive option with uncertain consequences and side effects, natural herbal therapy offers an attractive alternative. The purpose of this study is to evaluate the effect on BMD and safety of the Classic Yin and Yang Tonic Formula for treatment of osteopenia and to investigate the mechanism by which this efficacy is achieved., Methods/design: We propose a multicenter double-blind randomized placebo-controlled trial to evaluate the efficacy and safety of the Classic Yin and Yang Tonic Formula for the treatment of osteopenia. Participants aged 55 to 75 with low bone mineral density (T-score between -1 and -2.5) and kidney deficiency in TCM will be included and randomly allocated into two groups: treatment group and control group. Participants in the treatment group will be treated with Classic Yin and Yang Tonic Granule, while the controlled group will receive placebo. Primary outcome measure will be BMD of the lumbar spine and proximal femur using dual-energy X-ray absorptiometry. Secondary outcomes will include pain intensity measured with visual analogue scales, quality of life, serum markers of bone metabolism, indices of Neuro-endocrino-immune network and safety., Discussion: If the Classic Yin and Yang Tonic Formula can increase bone mass without adverse effects, it may be a novel strategy for the treatment of osteoporosis. Furthermore, the mechanism of the Chinese medical formula for osteoporosis will be partially elucidated., Trial Registration: This study is registered at ClinicalTrials.gov, NCT01271647.

Objective: Abnormal maturation and ossification of the endplate chondrocytes play a central role in the pathogenesis of degenerative disorders of the cervical spine. It is widely held that insulin like growth factor-1 (IGF-1) stimulates chondrocyte proliferation and inhibits chondrocyte terminal differentiation both in vitro and in vivo. However, the mechanism underlying such regulation is not fully understood. The present study aimed to determine the role of IGF-1 on the mRNA expression of collagen type II, alpha 1 (Col2a1) and matrix metallopeptidase 13 (MMP-13) in rat endplate chondrocytes. The possible pathways that transduce IGF-1 effects such as phosphatidylinositol-3 (PI-3)-kinase (PI3K) and mitogen activated protein kinase (MAPK) were also investigated in these cells., Methods: Cultured endplate chondrocytes harvested from rat cervical spines were treated with IGF-1 (100ng/ml), and the changes in Col2a1 and MMP-13 mRNA were monitored with real-time polymerase chain reaction (PCR). MMP-13 activity was also assayed. Activation of signaling proteins was evaluated by western blot analysis. Cells were also treated with pharmacological agents that block PI3K and MAPK signaling pathways., Results: IGF-1 increased Col2a1 mRNA expression in rat endplate chondrocytes in a time- and dose-dependent manner. IGF-1 treatment resulted in a fourfold increase of Col2a1 mRNA with the effect maximizing at 24h. In contrast, IGF-1 treatment for 24h caused a roughly 50% reduction in MMP-13 mRNA. Similar effects were seen on the protein levels of type II collagen (col2) and MMP-13. Consistent with these results, IGF-1 also repressed MMP-13 activity. IGF-1 activated both the PI3K and the extracellular signal-regulated kinase (ERK) pathways as evidenced by phosphorylation of either Akt or ERK1/2 (respectively). The PI3K inhibitor Wartmannin significantly inhibited the IGF-1 effect on Col2a1 mRNA expression but did not affect IGF-1-induced repression of MMP-13 expression. In contrast, the ERK/MAPK inhibitor PD98059 significantly inhibited the effect of IGF-1 on MMP-13 mRNA repression and enhanced IGF-1-induced Col2a1 mRNA expression., Conclusions: In rat endplate chondrocytes the PI3K pathway mainly transduces IGF-1 effect on col2 expression while the ERK pathway mediates IGF-1 effect on MMP-13 expression.

Unlabelled: The AHR mediates many of the toxicological effects of aromatic hydrocarbons. We show that AHR expression in osteoblasts parallels the induction of early bone-specific genes involved in maturation. The AHR may not only mediate the effects of toxicants, but with an as yet unidentified ligand, be involved in the differentiation pathways of osteoblasts., Introduction: Metabolic bone diseases arise as a result of an imbalance in bone cell activities. Recent evidence suggests that environmental toxicants may be contributing factors altering these activities. One candidate molecule implicated in mediating the toxic effects of exogenous compounds is the aryl hydrocarbon receptor (AHR)., Materials and Methods: Osteoblasts isolated from neonatal rat calvaria were analyzed for AHR expression by quantitative PCR, Western blot, and immunohistochemistry. In addition, AHR activation was evaluated by electromobility gel shift assay and fluorescence microscopy., Results: Our findings showed AHR expression in mature osteoblasts in vivo. The pattern of AHR expression peaks after alkaline phosphatase and before induction of osteocalcin. We first show that AHR functions as a transactivating receptor in osteoblasts, as evidenced by its ligand-dependent migration to the nucleus and its association with known dioxin response elements. AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) mediated the induction of cytochrome p450 1A1 and cycloxygenase-2 protein levels. This effect could be inhibited by the potent AHR antagonist, 3'4 methoxynitroflavone. Furthermore, lead treatment of osteoblasts upregulates the expression of AHR mRNA and protein levels, supporting a novel mechanism whereby lead in the skeleton may increase the sensitivity of bone cells to toxicant exposure., Conclusions: These data imply that the AHR mediates the effects of aromatic toxicants on bone and that AHR expression is regulated during osteoblast differentiation.

In this treatise we will examine complexities in the development and function of cells of the musculoskeletal system. Specifically, the role of chondrocytes and their ontogeny and osteoblasts and their ontogeny will be discussed as they regulate cartilage and bone formation. This background information will provide the foundation for evaluating the effects of environmental toxicants on skeletal development. A number of agents such as heavy metals (i.e. lead) and polycyclic aromatic hydrocarbons (i.e. pesticides and cigarette smoke) interact with cells of the skeletal system and adversely affect development. These agents have not been of major research interest, nevertheless, given changes in the environmental profile of the United States and other developed countries, it is important that we understand their effects in bone and cartilage. Research in this area will identify strategies that may be used to help prevent musculoskeletal diseases due to toxicant exposure., ((c) 2007 Wiley-Liss, Inc.)