Gap junctional communication promotes apoptosis in a connexin-type-dependent manner

Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to α-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca(2+) signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca(2+) concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca(2+) or a Ca(2+)-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca(2+) increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells

Loops versus lines and the compression stiffening of cells

Both animal and plant tissue exhibit a nonlinear rheological phenomenon known as compression stiffening, or an increase in moduli with increasing uniaxial compressive strain. Does such a phenomenon exist in single cells, which are the building blocks of tissues? One expects an individual cell to compression soften since the semiflexible biopolymer-based cytoskeletal network maintains the mechanical integrity of the cell and in vitro semiflexible biopolymer networks typically compression soften. To the contrary, we find that mouse embryonic fibroblasts (mEFs) compression stiffen under uniaxial compression via atomic force microscopy (AFM) studies. To understand this finding, we uncover several potential mechanisms for compression stiffening. First, we study a single semiflexible polymer loop modeling the actomyosin cortex enclosing a viscous medium modeled as an incompressible fluid. Second, we study a two-dimensional semiflexible polymer/fiber network interspersed with area-conserving loops, which are a proxy for vesicles and fluid-based organelles. Third, we study two-dimensional fiber networks with angular-constraining crosslinks, i.e. semiflexible loops on the mesh scale. In the latter two cases, the loops act as geometric constraints on the fiber network to help stiffen it via increased angular interactions. We find that the single semiflexible polymer loop model agrees well with our AFM experiments until approximately 35% compressive strain. We also find for the fiber network with area-conserving loops model that the stress-strain curves are sensitive to the packing fraction and size distribution of the area-conserving loops, thereby creating a mechanical fingerprint across different cell types. Finally, we make comparisons between this model and experiments on fibrin networks interlaced with beads as well as discuss the tissue-scale implications of cellular compression stiffening.Comment: 19 pages, 17 figure

High fluoride and low calcium levels in drinking water is associated with low bone mass, reduced bone quality and fragility fractures in sheep

SUMMARY: Chronic environmental fluoride exposure under calcium stress causes fragility fractures due to osteoporosis and bone quality deterioration, at least in sheep. Proof of skeletal fluorosis, presenting without increased bone density, calls for a review of fracture incidence in areas with fluoridated groundwater, including an analysis of patients with low bone mass. INTRODUCTION: Understanding the skeletal effects of environmental fluoride exposure especially under calcium stress remains an unmet need of critical importance. Therefore, we studied the skeletal phenotype of sheep chronically exposed to highly fluoridated water in the Kalahari Desert, where livestock is known to present with fragility fractures. METHODS: Dorper ewes from two flocks in Namibia were studied. Chemical analyses of water, blood and urine were executed for both cohorts. Skeletal phenotyping comprised micro-computer tomography (μCT), histological, histomorphometric, biomechanical, quantitative backscattered electron imaging (qBEI) and energy-dispersive X-ray (EDX) analysis. Analysis was performed in direct comparison with undecalcified human iliac crest bone biopsies of patients with fluoride-induced osteopathy. RESULTS: The fluoride content of water, blood and urine was significantly elevated in the Kalahari group compared to the control. Surprisingly, a significant decrease in both cortical and trabecular bones was found in sheep chronically exposed to fluoride. Furthermore, osteoid parameters and the degree and heterogeneity of mineralization were increased. The latter findings are reminiscent of those found in osteoporotic patients with treatment-induced fluorosis. Mechanical testing revealed a significant decrease in the bending strength, concurrent with the clinical observation of fragility fractures in sheep within an area of environmental fluoride exposure. CONCLUSIONS: Our data suggest that fluoride exposure with concomitant calcium deficit (i) may aggravate bone loss via reductions in mineralized trabecular and cortical bone mass and (ii) can cause fragility fractures and (iii) that the prevalence of skeletal fluorosis especially due to groundwater exposure should be reviewed in many areas of the world as low bone mass alone does not exclude fluorosis

Pinealectomy affects bone mineral density and structure - an experimental study in sheep

<p>Abstract</p> <p>Background</p> <p>Osteoporosis and associated fractures are a major public health burden and there is great need for a large animal model. Melatonin, the hormone of the pineal gland, has been shown to influence bone metabolism. This study aims to evaluate whether absence of melatonin due to pinealectomy affects the bone mass, structure and remodeling in an ovine animal model.</p> <p>Methods</p> <p>Female sheep were arranged into four groups: Control, surgically ovariectomized (Ovx), surgically pinealectomized (Px) and Ovx+Px. Before and 6 months after surgery, iliac crest biopsies were harvested and structural parameters were measured using μCT. Markers of bone formation and resorption were determined. To evaluate long term changes after pinealectomy, bone mineral density (BMD) was analyzed at the distal radius at 0, 3, 9, 18 and 30 months.</p> <p>Results</p> <p>Cancellous bone volume (BV/TV) declined after 6 months by -13.3% Px and -21.5% OvxPx. The bone loss was due to increased trabecular separation as well as decreased thickness. The histomorphometric quantification and determination of collagen degradation products showed increased bone resorption following pinealectomy. Ovariectomy alone results in a transient bone loss at the distal radius followed by continuous increase to baseline levels. The bone resorption activity after pinealectomy causes a bone loss which was not transient, since a continuous decrease in BMD was observed until 30 months.</p> <p>Conclusions</p> <p>The changes after pinealectomy in sheep are indicative of bone loss. Overall, these findings suggest that the pineal gland may influence bone metabolism and that pinealectomy can be used to induce bone loss in sheep.</p

Clinical behavior and outcomes of breast cancer in young women with germline BRCA pathogenic variants

Young breast cancer (BC) patients carrying a germline BRCA pathogenic variant (mBRCA) have similar outcomes as non-carriers. However, the impact of the type of gene (BRCA1 vs. BRCA2) and hormone receptor status (positive [HR+] vs. negative [HR 12]) on clinical behavior and outcomes of mBRCA BC remains largely unknown. This is an international, multicenter, hospital-based, retrospective cohort study that included mBRCA patients diagnosed, between January 2000 and December 2012, with stage I\u2013III invasive early BC at age 6440 years. From 30 centers worldwide, 1236 young mBRCA BC patients were included. Among 808 and 428 patients with mBRCA1 or mBRCA2, 191 (23.6%) and 356 (83.2%) had HR+tumors, respectively (P < 0.001). Median follow-up was 7.9 years. Second primary BC (P = 0.009) and non-BC malignancies (P = 0.02) were more frequent among mBRCA1 patients while distant recurrences were less frequent (P = 0.02). Irrespective of hormone receptor status, mBRCA1 patients had worse disease-free survival (DFS; adjusted HR = 0.76, 95% CI = 0.60\u20130.96), with no difference in distant recurrence-free interval (DRFI) and overall survival (OS). Patients with HR+ disease had more frequent distant recurrences (P < 0.001) and less frequent second primary malignancies (BC: P = 0.005; non-BC: P = 0.18). No differences in DFS and OS were observed according to hormone receptor status, with a tendency for worse DRFI (adjusted HR = 1.39, 95% CI = 0.94\u20132.05) in patients with HR+ BC. Type of mBRCA gene and hormone receptor status strongly impact BC clinical behavior and outcomes in mBRCA young patients. These results provide important information for patients\u2019 counseling on treatment, prevention, and surveillance strategies

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats

Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P < 0.001). The number of TRAP+ osteoclasts in bone resorption pits, VEGF+ cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P < 0.05), while no significant difference was detected in the number of ALP+ cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system