Lysosomal exocytosis: From cell protection to protumoral functions.

Lysosomes are single membrane bounded group of acidic organelles that can be involved in a process called lysosomal exocytosis which leads to the extracellular release of their content. Lysosomal exocytosis is required for plasma membrane repair or remodeling events such as bone resorption, antigen presentation or mitosis, and for protection against toxic agents such as heavy metals. Recently, it has been showed that to fulfill this protective role, lysosomal exocytosis needs some autophagic proteins, in an autophagy-independent manner. In addition to these crucial physiological roles, lysosomal exocytosis plays a major protumoral role in various cancers. This effect is exerted through tumor microenvironment modifications, including extracellular matrix remodeling, acidosis, oncogenic and profibrogenic signals. This review provides a comprehensive overview of the different elements released in the microenvironment during lysosomal exocytosis, i.e. proteases, exosomes, and protons, and their effects in the context of tumor development and treatment.

Comparison of false starts by saw created on flesh and dry bones; as close as possible to the real conditions of criminal dismemberment.

In most experimental protocols, false starts are produced on dry bones obtained through a maceration process for anthropological analyses, for the sake of reproducibility. Although this allows for controlled experimental conditions, the absence of soft parts when experimentally creating false starts does not correspond to the real conditions of criminal dismemberment. The main objective of this study was to determine if the results of experimental work on the characteristics of false starts were valid under medico-legal conditions. In this experimental study, a hand saw (rip saw, wavy set, TPI 32) was used. 240 false starts were produced on human and pig bones. Randomly, the false starts were either produced on a dry bone or on a flesh bone. The criteria for microscopic analysis included the shape of the walls, the shape and visibility of striae on the floor, the shape of the profile, and the minimum width of the false start. On human bone, 100% of the false starts produced on a bone that had previously undergone a maceration process for anthropological analyses (dry bone) allowed the definition of all the blade characteristics. This was the case for 78.3% on bone in the presence of soft tissue (flesh bone). The striae on the floor of the false start are in some cases less visible with flesh bones, implying that it may be more difficult to conclude on the characteristics of a saw under medico-legal conditions.

Autophagy markers are decreased in bone of osteoporotic patients: a monocentric comparative study.

BACKGROUND: Osteoporosis (OP) is a pathology characterized by bone fragility affecting 30% of postmenopausal women, mainly due to estrogen deprivation and increased oxidative stress. An autophagy involvement is suspected in OP pathogenesis but a definitive proof in humans remains to be obtained. METHODS: Postmenopausal women hospitalized for femoral neck fracture (OP group) or total hip replacement (Control group) were enrolled using very strict exclusion criteria. Western blot was used to analyze autophagy level. RESULTS: The protein expression level of the autophagosome marker LC3-II was significantly decreased in bone of OP patients relative to the control group. In addition, the protein expression of the hormonally upregulated neu-associated kinase (HUNK), which is upregulated by female hormones and promotes autophagy, was also significantly reduced in bone of the OP group. CONCLUSIONS: These results demonstrate for the first time that postmenopausal OP patients have a deficit in bone autophagy level and suggest that HUNK could be the factor linking estrogen loss and autophagy decline. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT03175874, 2/6/2017.

Comparison of microscopic features of bone false starts produced by reciprocating saw blades and handsaws of the same class.

In cases of criminal dismemberment, the analysis of saw marks helps to determine the class of the saw used. The present study compared the microscopic features of experimental false starts performed on three freshly defleshed human femurs. 150 lesions were produced using three reciprocating blades and two hand saws of similar class. Two groups of tools were created according to the class of the blades: group 1 (teeth per inch = 6, alternating set) and group 2 (teeth per inch = 24, wavy set). The minimum width of the kerf was always thicker with reciprocating blades than that of the handsaw of the same class. This fact is linked to the thickness of the blades and not necessarily to the fast forth and back motion of the blades. Shape kerf profiles and shape of the walls were very confusing, with a lot of variation of the false starts caused by the reciprocating blades and the risk of misclassification (rip teeth versus crosscut teeth, alternating set versus wavy set). Striae on the kerf floor and bone islands were more consistent. The main conclusions are, first, that usual features of false starts achieved by reciprocating saw blades used in this work vary a great deal and may lead to misclassification of the saw class, and second, that no criterion has been found to differentiate reciprocating blades and handsaws. These results demonstrate that we have to be very prudent while estimating the possible saw from a false start in a real forensic case.

Chelating Polymers for Targeted Decontamination of Actinides: Application of PEI-MP to Hydroxyapatite-Th(IV).

In case of an incident in the nuclear industry or an act of war or terrorism, the dissemination of plutonium could contaminate the environment and, hence, humans. Human contamination mainly occurs via inhalation and/or wounding (and, less likely, ingestion). In such cases, plutonium, if soluble, reaches circulation, whereas the poorly soluble fraction (such as small colloids) is trapped in alveolar macrophages or remains at the site of wounding. Once in the blood, the plutonium is delivered to the liver and/or to the bone, particularly into its mineral part, mostly composed of hydroxyapatite. Countermeasures against plutonium exist and consist of intravenous injections or inhalation of diethylenetetraminepentaacetate salts. Their effectiveness is, however, mainly confined to the circulating soluble forms of plutonium. Furthermore, the short bioavailability of diethylenetetraminepentaacetate results in its rapid elimination. To overcome these limitations and to provide a complementary approach to this common therapy, we developed polymeric analogs to indirectly target the problematic retention sites. We present herein a first study regarding the decontamination abilities of polyethyleneimine methylcarboxylate (structural diethylenetetraminepentaacetate polymer analog) and polyethyleneimine methylphosphonate (phosphonate polymeric analog) directed against Th(IV), used here as a Pu(IV) surrogate, which was incorporated into hydroxyapatite used as a bone model. Our results suggest that polyethylenimine methylphosphonate could be a good candidate for powerful bone decontamination action.

Autophagy and bone diseases.

Autophagy is a ubiquitous cellular process, allowing the removal and recycling of damaged proteins and organelles. At the basal level, this process plays a role in quality control, thus participating in cellular homeostasis. Autophagy can also be induced by various stresses, such as nutrient deprivation or hypoxia, to allow the cell to survive until conditions improve. In recent years, the role of this process has been widely studied in many pathologies such as neurodegenerative diseases or cancers. In bone tissue, various studies have shown that autophagy is involved in the survival, differentiation and activity of osteoblasts, osteocytes and osteoclasts. The evolution of this knowledge has led to the identification of new molecular pathophysiological mechanisms in bone pathologies. This review reports the current state of knowledge on the role of autophagy in 4 bone diseases: osteoporosis, which seems to be associated with a decrease in autophagy, osteopetrosis and Paget's disease where the course of the autophagic process is disturbed, and finally osteosarcoma where autophagy seems to play a protumoral role. A better understanding of the involvement of autophagy in these pathologies should eventually lead to the identification of new potential therapeutic targets.

The stereomicroscope and scanning electron microscope comparison of false starts bones produced by an oscillating autopsy saw.

In forensic anthropology, saw mark analysis plays an important role in cases of criminal dismemberment. Autopsy saw is not used by the perpetrator in cases of dismemberment, but the forensic pathologist may accidentally create false starts with this saw during an autopsy, especially while sampling bones for further analysis, and these autopsy false starts can be confused with false starts produced by the offender. In this study, the characteristics of 20 false starts were compared using stereomicroscopy (SM) and scanning electron microscopy (SEM). These bone false starts were selected at random from a previous study of 100 false starts created by an electrical oscillating autopsy saw on human femoral bones. That study had enabled the categorization of the lesions into two groups ("superficial group" and "deep group") with a 0.52 mm depth cut off, based on the dramatic differences in lesion characteristics between these two groups. In the current study, SEM confirmed the characteristics of the false starts (walls and profile shapes, striae, bone islands and bone debris were studied), and above all explained the mechanism whereby oval bone islands in deep lesions are formed. Bone islands are due to the horizontal and vertical movement of the oscillating autopsy blade.

Low doses of uranium and osteoclastic bone resorption: key reciprocal effects evidenced using new in vitro biomimetic models of bone matrix.

Uranium is widely spread in the environment due to its natural and anthropogenic occurrences, hence the importance of understanding its impact on human health. The skeleton is the main site of long-term accumulation of this actinide. However, interactions of this metal with biological processes involving the mineralized extracellular matrix and bone cells are still poorly understood. To get a better insight into these interactions, we developed new biomimetic bone matrices containing low doses of natural uranium (up to 0.85 µg of uranium per cm2). These models were characterized by spectroscopic and microscopic approaches before being used as a support for the culture and differentiation of pre-osteoclastic cells. In doing so, we demonstrate that uranium can exert opposite effects on osteoclast resorption depending on its concentration in the bone microenvironment. Our results also provide evidence for the first time that resorption contributes to the remobilization of bone matrix-bound uranium. In agreement with this, we identified, by HRTEM, uranium phosphate internalized in vesicles of resorbing osteoclasts. Thanks to the biomimetic matrices we developed, this study highlights the complex mutual effects between osteoclasts and uranium. This demonstrates the relevance of these 3D models to further study the cellular mechanisms at play in response to uranium storage in bone tissue, and thus better understand the impact of environmental exposure to uranium on human bone health.

Autophagy in Osteosarcoma Cancer Stem Cells Is Critical Process which Can Be Targeted by the Antipsychotic Drug Thioridazine.

Cancer stem cells (CSCs) represent a minor population of cancer cells with stem cell-like properties which are able to fuel tumor growth and resist conventional treatments. Autophagy has been described to be upregulated in some CSCs and to play a crucial role by maintaining stem features and promoting resistance to both hostile microenvironments and treatments. Osteosarcoma (OS) is an aggressive bone cancer which mainly affects children and adolescents and autophagy in OS CSCs has been poorly studied. However, this is a very interesting case because autophagy is often deregulated in this cancer. In the present work, we used two OS cell lines showing different autophagy capacities to isolate CSC-enriched populations and to analyze the autophagy in basal and nutrient-deprived conditions. Our results indicate that autophagy is more efficient in CSCs populations compared to the parental cell lines, suggesting that autophagy is a critical process in OS CSCs. We also showed that the antipsychotic drug thioridazine is able to stimulate, and then impair autophagy in both CSC-enriched populations, leading to autosis, a cell death mediated by the Na+/K+ ATPase pump and triggered by dysregulated accumulation of autophagosomes. Taken together, our results indicate that autophagy is very active in OS CSCs and that targeting this pathway to switch their fate from survival to death could provide a novel strategy to eradicate these cells in osteosarcoma.

Autophagy in the crosstalk between tumor and microenvironment.

Autophagy is the major catabolic process in eukaryotic cells for the degradation and recycling of damaged macromolecules and organelles. It plays a crucial role in cell quality control and nutrient supply under stress conditions. Although autophagy is classically described as a degradative mechanism, it can also be involved in some secretion pathways, leading to the extracellular release of proteins, aggregates, or organelles. The role of autophagy in cancer is complex and depends on tumor development stage. While autophagy limits cancer development in the early stages of tumorigenesis, it can also have a protumoral role in more advanced cancers, promoting primary tumor growth and metastatic spread. In addition to its pro-survival role in established tumors, autophagy recently emerged as an active player in the crosstalk between tumor and stromal cells. The aim of this review is to analyze the impact of tumoral autophagy on the microenvironment and conversely the effect of stromal cell autophagy on tumor cells.

Uranium Effect on Osteocytic Cells In Vitro.

Once absorbed in the body, natural uranium [U(VI)], a radionucleotide naturally present in the environment, is targeted to the skeleton which is the long-term storage organ. We and others have reported the U(VI) negative effects on osteoblasts (OB) and osteoclasts (OC), the main two cell types involved in bone remodeling. In the present work, we addressed the U(VI) effect on osteocytes (OST), the longest living bone cell type and the more numerous (> 90%). These cells, which are embedded in bone matrix and thus are the more prone to U(VI) long-term exposure, are now considered as the chief orchestrators of the bone remodeling process. Our results show that the cytotoxicity index of OST is close to 730 µM, which is about twice the one reported for OB and OC. However, despite this resistance potential, we observed that chronic U(VI) exposure as low as 5 µM led to a drastic decrease of the OST mineralization function. Gene expression analysis showed that this impairment could potentially be linked to an altered differentiation process of these cells. We also observed that U(VI) was able to trigger autophagy, a highly conserved survival mechanism. Extended X-ray absorption fine structure analysis at the U LIII edge of OST cells exposed to U(VI) unambiguously shows the formation of an uranyl phosphate phase in which the uranyl local structure is similar to the one present in Autunite. Thus, our results demonstrate for the first time that OST mineralization function can be affected by U(VI) exposure as low as 5 µM, suggesting that prolonged exposure could alter the central role of these cells in the bone environment.

Role of autophagy in osteosarcoma.

Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. It is a highly aggressive tumor with a tendency to spread to the lungs, which are the most common site of metastasis. Advanced osteosarcoma patients with metastasis share a poor prognosis. Despite the use of chemotherapy to treat OS, the 5-year overall survival rate for patients has remained unchanged at 65-70% for the past 20 years. In addition, the 5-year survival of patients with a metastatic disease is around 20%, highlighting the need for novel therapeutic targets. Autophagy is an intracellular degradation process which eliminates and recycles damaged proteins and organelles to improve cell lifespan. In the context of cancer, numerous studies have demonstrated that autophagy is used by tumor cells to repress initial steps of carcinogenesis and/or support the survival and growth of established tumors. In osteosarcoma, autophagy appears to be deregulated and could also act both as a pro or anti-tumoral process. In this manuscript, we aim to review these major findings regarding the role of autophagy in osteosarcoma.

Natural uranium impairs the differentiation and the resorbing function of osteoclasts.

BACKGROUND: Uranium is a naturally occurring radionuclide ubiquitously present in the environment. The skeleton is the main site of uranium long-term accumulation. While it has been shown that natural uranium is able to perturb bone metabolism through its chemical toxicity, its impact on bone resorption by osteoclasts has been poorly explored. Here, we examined for the first time in vitro effects of natural uranium on osteoclasts. METHODS: The effects of uranium on the RAW 264.7 monocyte/macrophage mouse cell line and primary murine osteoclastic cells were characterized by biochemical, molecular and functional analyses. RESULTS: We observed a cytotoxicity effect of uranium on osteoclast precursors. Uranium concentrations in the µM range are able to inhibit osteoclast formation, mature osteoclast survival and mineral resorption but don't affect the expression of the osteoclast gene markers Nfatc1, Dc-stamp, Ctsk, Acp5, Atp6v0a3 or Atp6v0d2 in RAW 274.7 cells. Instead, we observed that uranium induces a dose-dependent accumulation of SQSTM1/p62 during osteoclastogenesis. CONCLUSIONS: We show here that uranium impairs osteoclast formation and function in vitro. The decrease in available precursor cells, as well as the reduced viability of mature osteoclasts appears to account for these effects of uranium. The SQSTM1/p62 level increase observed in response to uranium exposure is of particular interest since this protein is a known regulator of osteoclast formation. A tempting hypothesis discussed herein is that SQSTM1/p62 dysregulation contributes to uranium effects on osteoclastogenesis. GENERAL SIGNIFICANCE: We describe cellular and molecular effects of uranium that potentially affect bone homeostasis.

Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.

Natural uranium (U), which is present in our environment, exerts a chemical toxicity, particularly in bone where it accumulates. Generally, U is found at oxidation state +VI in its oxocationic form [Formula: see text] in aqueous media. Although U(VI) has been reported to induce cell death in osteoblasts, the cells in charge of bone formation, the molecular mechanism for U(VI) effects in these cells remains poorly understood. The objective of our study was to explore U(VI) effect at doses ranging from 5 to 600 µM, on mineralization and autophagy induction in the UMR-106 model osteoblastic cell line and to determine U(VI) speciation after cellular uptake. Our results indicate that U(VI) affects mineralization function, even at subtoxic concentrations (<100 µM). The combination of thermodynamic modeling of U with EXAFS data in the culture medium and in the cells clearly indicates the biotransformation of U(VI) carbonate species into a meta-autunite phase upon uptake by osteoblasts. We next assessed U(VI) effect at 100 and 300 µM on autophagy, a survival process triggered by various stresses such as metal exposure. We observed that U(VI) was able to rapidly activate autophagy but an inhibition of the autophagic flux was observed after 24 h. Thus, our results indicate that U(VI) perturbs osteoblastic functions by reducing mineralization capacity. Our study identifies for the first time U(VI) in the form of meta-autunite in mammalian cells. In addition, U(VI)-mediated inhibition of the autophagic flux may be one of the underlying mechanisms leading to the decreased mineralization and the toxicity observed in osteoblasts.

Sex-specific autophagy modulation in osteoblastic lineage: a critical function to counteract bone loss in female.

Age-related bone loss is associated with an increased oxidative stress which is worsened by estrogen fall during menauposis. This observation has drawn attention to autophagy, a major cellular catabolic process, able to alleviate oxidative stress in osteoblasts (OB) and osteocytes (OST), two key bone cell types. Moreover, an autophagy decline can be associated with aging, suggesting that an age-related autophagy deficiency in OB and/or OST could contribute to skeletal aging and osteoporosis onset.In the present work, autophagy activity was analyzed in OST and OB in male and female mice according to their age and hormonal status. In OST, autophagy decreases with aging in both sexes. In OB, although a 95% decrease in autophagy is observed in OB derived from old females, this activity remains unchanged in males. In addition, while ovariectomy has no effect on OB autophagy levels, orchidectomy appears to stimulate this process. An inverse correlation between autophagy and the oxidative stress level was observed in OB derived from males or females. Finally, using OB-specific autophagy-deficient mice, we showed that autophagy deficiency aggravates the bone loss associated with aging and estrogen deprivation.Taken together, our data indicate that autophagic modulation in bone cells differs according to sex and cell type. The lowering of autophagy in female OB, which is associated with an increased oxidative stress, could play a role in osteoporosis pathophysiology and suggests that autophagy could be a new therapeutic target for osteoporosis in women.

Pelvic fractures: epidemiology, consequences, and medical management.

PURPOSE OF REVIEW: Until recently, osteoporotic pelvic fractures have not been specifically studied. This review presents an update on epidemiological data of pelvic fracture, including morbidity, mortality and healthcare costs, the role of surgery and new data on sacroplasty in acute phase management. RECENT FINDINGS: All studies underline the burden of osteoporotic pelvic fractures. Risk factors associated with these fractures are age, sex (women), and previous loss of autonomy. An increased mortality has been reported in all publications, similar to hip fracture for in-patient mortality and at 5 years of follow-up. Pelvic fractures often lead to transient or permanent autonomy loss, reflecting the high costs because of extended hospital stay, combined with nursing home requirement. However, recent studies report a decrease in the length of stay. Sacroplasty displays promising results to control pain and improve functional outcome. Early surgery begins to be discussed to also improve the outcome. SUMMARY: Pelvic fractures display all the features of severe osteoporotic fractures: increased incidence, high morbidity, mortality, and healthcare costs that justify awareness of the practitioner on these fractures. Further studies on sacroplasty and surgery are necessary to improve pain control, functional improvement, thereby reducing the length of hospital stay and cost.

Autophagy in bone: Self-eating to stay in balance.

Autophagy, a major catabolic pathway responsible of the elimination of damaged proteins and organelles, is now recognized as an anti-aging process. In addition to its basal role in cell homeostasis, autophagy is also a stress-responsive mechanism for survival purposes. Here, we review recent literature to highlight the autophagy role in the different bone cell types, i.e., osteoblasts, osteoclasts and osteocytes. We also discuss the effects of autophagy modulators in bone physiology and of bone anabolic compounds in autophagy. Finally, we analyzed studies regarding bone cell autophagy-deficient mouse models to obtain a more general view on how autophagy modulates bone physiology and pathophysiology, particularly during aging.

Autophagy in osteoblasts is involved in mineralization and bone homeostasis.

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.