Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

Background

Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues.

Results

In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis.

Conclusions

Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.     

The lack of ADAM17 activity during embryonic development causes hemorrhage and impairs vessel formation

Background

ADAM17/TACE activity is important during embryonic development. We wished to investigate possible roles of this metalloprotease, focusing on vascular development.

Methodology/Principal Findings

Mice mutant in the enzymatic activity of ADAM17 were examined at various stages of embryonic development for vascular pattern and integrity using markers for vessel wall cells. We observed hemorrhage and edema starting at embryonic day E14.5 and becoming more severe as development proceeded; prior to embryonic day E14.5, embryos appeared normal. Staining for PECAM-1/CD31 revealed abnormalities in the patterns of branching of the embryonic vasculature at E14.5.

Conclusions/Significance

These abnormalities preceded association of pericytes or monocyte/macrophage cells with the affected vessels and, therefore, presumably arise from defects in endothelial function consequent upon failure of ADAM17 to cleave one or more substrates involved in vascular development, such as Notch, Delta, VEGFR2 or JAM-A. Our study demonstrates a role for ADAM17 in modulating embryonic vessel development and function.     

A New Metabolomic Signature in Type-2 Diabetes Mellitus and Its Pathophysiology

Objective

The objective of the current study was to find a metabolic signature associated with the early manifestations of type-2 diabetes mellitus.

Research Design and Method

Modern metabolic profiling technology (MxP™ Broad Profiling) was applied to find early alterations in the plasma metabolome of type-2 diabetic patients. The results were validated in an independent study. Eicosanoid and single inon monitoring analysis (MxP™ Eicosanoid and MxP™ SIM analysis) were performed in subsets of samples.

Results

A metabolic signature including significantly increased levels of glyoxylate as a potential novel marker for early detection of type-2 diabetes mellitus was identified in an initial study (Study1). The signature was significantly altered in fasted diabetic and pre-diabetic subjects and in non-fasted subjects up to three years prior to the diagnosis of type-2 diabetes; most alterations were also consistently found in an independent patient group (Study 2). In Study 2 diabetic and most control subjects suffered from heart failure. In Study 1 a subgroup of diabetic subjects, with a history of use of anti-hypertensive medication further showed a more pronounced increase of glyoxylate levels, compared to a non-diabetic control group when tested in a hyperglycemic state. In the context of a prior history of anti-hypertensive medication, alterations in hexosamine and eicosanoid levels were also found.

Conclusion

A metabolic signature including glyoxylate was associated with type-2 diabetes mellitus, independent of the fasting status and of occurrence of another major disease. The same signature was also found to be associated with pre-diabetic subjects. Glyoxylate levels further showed a specifically strong increase in a subgroup of diabetic subjects. It could represent a new marker for the detection of medical subgroups of diabetic subjects.     

Serum Level of Soluble Receptor for Advanced Glycation End Products Is Associated with A Disintegrin And Metalloproteinase 10 in Type 1 Diabetes

Background

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of diabetic complications, and soluble forms of the receptor (sRAGE) can counteract the detrimental action of the full-length receptor by acting as decoy. Soluble RAGE is produced by alternative splicing [endogenous secretory RAGE (esRAGE)] and/or by proteolytic cleavage of the membrane-bound receptor. We have investigated the role of A Disintegrin And Metalloproteinase 10 (ADAM10) in the ectodomain shedding of RAGE.

Methods

Constitutive and insulin-induced shedding of RAGE in THP-1 macrophages by ADAM10 was evaluated using an ADAM10-specific metalloproteinase inhibitor. Serum ADAM10 level was measured in type 1 diabetes and control subjects, and the association with serum soluble RAGE was determined. Serum total sRAGE and esRAGE were assayed by ELISA and the difference between total sRAGE and esRAGE gave an estimated measure of soluble RAGE formed by cleavage (cRAGE).

Results

RAGE shedding (constitutive and insulin-induced) was significantly reduced after inhibition of ADAM10 in macrophages, and insulin stimulated ADAM10 expression and activity. Diabetic subjects have higher serum total sRAGE and esRAGE (p<0.01) than controls, and serum ADAM10 was also increased (p<0.01). Serum ADAM10 correlated with serum cRAGE in type 1 diabetes (r = 0.40, p<0.01) and in controls (r = 0.31. p<0.01) but no correlations were seen with esRAGE. The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.

Conclusion

Our data suggested that ADAM10 contributed to the shedding of RAGE. Serum ADAM10 level was increased in type 1 diabetes and was a significant determinant of circulating cRAGE.     

Conceptualizing Community Mobilization for HIV Prevention: Implications for HIV Prevention Programming in the African Context

Introduction

Community mobilizing strategies are essential to health promotion and uptake of HIV prevention. However, there has been little conceptual work conducted to establish the core components of community mobilization, which are needed to guide HIV prevention programming and evaluation.

Objectives

We aimed to identify the key domains of community mobilization (CM) essential to change health outcomes or behaviors, and to determine whether these hypothesized CM domains were relevant to a rural South African setting.

Method

We studied social movements and community capacity, empowerment and development literatures, assessing common elements needed to operationalize HIV programs at a community level. After synthesizing these elements into six essential CM domains, we explored the salience of these CM domains qualitatively, through analysis of 10 key informant in-depth-interviews and seven focus groups in three villages in Bushbuckridge.

Results

CM domains include: 1) shared concerns, 2) critical consciousness, 3) organizational structures/networks, 4) leadership (individual and/or institutional), 5) collective activities/actions, and 6) social cohesion. Qualitative data indicated that the proposed domains tapped into theoretically consistent constructs comprising aspects of CM processes. Some domains, extracted from largely Western theory, required little adaptation for the South African context; others translated less effortlessly. For example, critical consciousness to collectively question and resolve community challenges functioned as expected. However, organizations/networks, while essential, operated differently than originally hypothesized - not through formal organizations, but through diffuse family networks.

Conclusions

To date, few community mobilizing efforts in HIV prevention have clearly defined the meaning and domains of CM prior to intervention design. We distilled six CM domains from the literature; all were pertinent to mobilization in rural South Africa. While some adaptation of specific domains is required, they provide an extremely valuable organizational tool to guide CM programming and evaluation of critically needed mobilizing initiatives in Southern Africa.     

Requirement of NOX2 Expression in Both Retina and Bone Marrow for Diabetes-Induced Retinal Vascular Injury

Objective

Diabetic retinopathy, a major cause of blindness, is characterized by increased expression of vascular endothelial growth factor (VEGF), leukocyte attachment to the vessel walls and increased vascular permeability. Previous work has shown that reactive oxygen species (ROS) produced by the superoxide generating enzyme NOX2/NADPH oxidase play a crucial role in the vascular pathology. The aim of this work was to identify the cellular sources of the damaging NOX2 activity by studies using bone marrow chimera mice.

Methods

Bone marrow cells were collected from the femurs and tibias of wild type and NOX2 deficient (NOX2^-/-) donor mice and injected intravenously into lethally irradiated NOX2^-/- and wild type recipients. Following recovery from radiation, mice were rendered diabetic by streptozotocin injections. The following groups of bone marrow chimeras were studied: non-diabetic WT→WT, diabetic WT→WT, diabetic WT→NOX2^-/-, diabetic NOX2^-/-→WT. After 4 weeks of diabetes, early signs of retinopathy were examined by measuring ROS, expression of VEGF and ICAM-1, leukocyte attachment to the vessel wall and vascular permeability.

Results

The retinas of the diabetic WT→WT chimeras showed significant increases in ROS as compared with the non-diabetic chimeras. These diabetes-induced alterations were correlated with increases in expression of VEGF and ICAM-1, leukocyte adhesion and vascular permeability. Each of these diabetes-induced alterations were significantly attenuated in the diabetic WT→NOX2^-/- and NOX2^-/-→WT chimera groups (p<0.05).

Conclusion

NOX2-generated ROS produced by both bone marrow-derived cells and resident retinal cells contribute importantly to retinal vascular injury in the diabetic retina. Targeting NOX2 in bone marrow and/or retinal cells may represent a novel therapeutic strategy for the treatment/prevention of vascular injury in the diabetic retina.     

A novel inhibitor of ADAM17 sensitizes colorectal cancer cells to 5‐Fluorouracil by reversing Notch and epithelial‐mesenchymal transition in vitro and in vivo

Objectives

Colorectal cancer is one of the most common malignancies both in men and women. Owing to metastasis and resistance, the prognosis of colorectal cancerCRC patients remains extremely poor with chemotherapy. A disintegrin and metalloproteinase 17 (ADAM17) induces the activation of Notch pathway and contributes to the chemoresistance. This study aimed to discover a novel ADAM17 inhibitor and investigate the chemosensitization effect.

Materials and methods

Pharmacophore model, western blot and enzymatic assay were used to discover ZLDI‐8. Cell proliferation was determined by MTT and colony formation assay. Cell migratory and invasive ability were determined by wound healing scratch and transwell assay. Immunofluorescence images and western blot analysed the expression of Notch or epithelial‐mesenchymal transition (EMT) pathway markers. Xenografts were employed to evaluate the chemosensitization effect of ZLDI‐8 in vivo.

Results

We found that ZLDI‐8 cell‐specifically inhibited the proliferation of CRC, and this effect was due to abrogation of ADAM17 and Notch pathway. Meanwhile, we reported for the first time that ZLDI‐8 synergistically improved the anti‐tumour and anti‐metastasis activity of 5‐fluorouracil or irinotecan by reversing Notch and EMT pathways. Interestingly, in vivo studies further demonstrated that ZLDI‐8 promoted the anti‐tumour effect of 5‐fluorouracil through Notch and EMT reversal.

Conclusions

A novel ADAM17 inhibitor ZLDI‐8 may be a potential chemosensitizer which sensitized CRC cells to 5‐fluorouracil or irinotecan by reversing Notch and EMT pathways.

     

Bronchial Smooth Muscle Cells of Asthmatics Promote Angiogenesis through Elevated Secretion of CXC-Chemokines (ENA-78, GRO-α, and IL-8)

Background

Airway wall remodelling is a key pathology of asthma. It includes thickening of the airway wall, hypertrophy and hyperplasia of bronchial smooth muscle cells (BSMC), as well as an increased vascularity of the sub-epithelial cell layer. BSMC are known to be the effector cells of bronchoconstriction, but they are increasingly recognized as an important source of inflammatory mediators and angiogenic factors.

Objective

To compare the angiogenic potential of BSMC of asthmatic and non-asthmatic patients and to identify asthma-specific angiogenic factors.

Methods

Primary BSMC were isolated from human airway tissue of asthmatic and non-asthmatic patients. Conditioned medium (CM) collected from BSMC isolates was tested for angiogenic capacity using the endothelial cell (EC)-spheroid in vitro angiogenesis assay. Angiogenic factors in CM were quantified using a human angiogenesis antibody array and enzyme linked immunosorbent assay.

Results

Induction of sprout outgrowth from EC-spheroids by CM of BSMC obtained from asthma patients was increased compared with CM of control BSMC (twofold, p < 0.001). Levels of ENA-78, GRO-α and IL-8 were significantly elevated in CM of BSMC from asthma patients (p < 0.05 vs. non-asthmatic patients). SB 265610, a competitive antagonist of chemokine (CXC-motif) receptor 2 (CXCR2), attenuated the increased sprout outgrowth induced by CM of asthma patient-derived BSMC.

Conclusions

BSMC isolated from asthma patients exhibit increased angiogenic potential. This effect is mediated through the CXCR2 ligands (ENA78, GRO-α and IL-8) produced by BSMC.

Implications

CXCR2 ligands may play a decisive role in directing the neovascularization in the sub-epithelial cell layers of the lungs of asthma patients. Counteracting the CXCR2-mediated neovascularization by pharmaceutical compounds may represent a novel strategy to reduce airway remodelling in asthma.     

Role of Endothelial Progenitor Cells and Inflammatory Cytokines in Healing of Diabetic Foot Ulcers

Background

To evaluate changes in endothelial progenitor cells (EPCs) and cytokines in patients with diabetic foot ulceration (DFU) in association with wound healing.

Methods

We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.

Results

All EPC phenotypes except the kinase insert domain receptor (KDR)⁺CD133⁺ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1) and stem cell factor (SCF) were increased in DFU patients. DFU patients who healed their ulcers had lower CD34⁺KDR⁺ count at visits 3 and 4, serum c-reactive protein (CRP) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at visit 1, interleukin-1 (IL-1) at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.

Conclusions

Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34⁺KDR⁺ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.     

Perivascular Delivery of Notch 1 siRNA Inhibits Injury-Induced Arterial Remodeling

Objectives

To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling.

Methods and Results

Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-X_L expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown.

Conclusion

These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.     

Deficiency of the Metalloproteinase-Disintegrin ADAM8 Is Associated with Thymic Hyper-Cellularity

Background

Thymopoiesis requires thymocyte-stroma interactions and proteases that promote cell migration by degrading extracellular matrix and releasing essential cytokines and chemokines. A role for several members of the A Disintegrin and Metalloprotease (ADAM) family in T cell development has been reported in the past.

Methodology/Principal Findings

Here, we present data indicating that the family member ADAM8 plays a role in thymic T cell development. We used qrtPCR on FACS sorted thymic subsets together with immunofluorescene to analyze thymic ADAM8 expression. We found that ADAM8 was expressed in murine thymic stromal cells and at lower levels in thymocytes where its expression increased as cell matured, suggesting involvement of ADAM8 in thymopoiesis. Further flow cytometry analysis revealed that ADAM8 deficient mice showed normal development and expansion of immature thymocyte subsets. There was however an intrathymic accumulation of single positive CD4 and CD8 T cells which was most noticeable in the late mature T cell subsets. Accumulation of single positive T cells coincided with changes in the thymic architecture manifest in a decreased cortex/medulla ratio and an increase in medullary epithelial cells as determined by histology and flow cytometry. The increase in single positive T cells was thymus-intrinsic, independent of progenitor homing to the thymus or thymic exit rate of mature T cells. Chemotaxis assays revealed that ADAM8 deficiency was associated with reduced migration of single positive thymocytes towards CCL21.

Conclusions/Significance

Our results show that ADAM8 is involved in T cell maturation in the medulla and suggest a role for this protease in fine-tuning maturation of thymocytes in the medulla. In contrast to ADAM10 and ADAM17 lack of ADAM8 appears to have a relatively minor impact on T cell development, which was unexpected given that maturation of thymocytes is dependent on proper localization and timing of migration.     

TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals

The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.     

Sunny Holidays before and after Melanoma Diagnosis Are Respectively Associated with Lower Breslow Thickness and Lower Relapse Rates in Italy

Background

Previous studies have reported an association between sun exposure and improved cutaneous melanoma (CM) survival. We analysed the association of UV exposure with prognostic factors and outcome in a large melanoma cohort.

Methods

A questionnaire was given to 289 (42%) CM patients at diagnosis (Group 1) and to 402 CM patients (58%) during follow-up (Group 2). Analyses were carried out to investigate the associations between sun exposure and melanoma prognostic factors and survival.

Results

Holidays in the sun two years before CM diagnosis were significantly associated with lower Breslow thickness (p=0.003), after multiple adjustment. Number of weeks of sunny holidays was also significantly and inversely associated with thickness in a dose-dependent manner (p=0.007). However when stratifying by gender this association was found only among women (p=0.0004) the risk of CM recurrence in both sexes was significantly lower in patients (n=271) who had holidays in the sun after diagnosis, after multiple adjustment including education: HR=0.30 (95%CI:0.10-0.87; p=0.03) conclusions: Holidays in the sun were associated with thinner melanomas in women and reduced rates of relapse in both sexes. However, these results do not prove a direct causal effect of sun exposure on survival since other confounding factors, such as vitamin D serum levels and socio-economic status, may play a role. Other factors in sun seeking individuals may also possibly affect these results.     

Rhinovirus-16 Induced Release of IP-10 and IL-8 Is Augmented by Th2 Cytokines in a Pediatric Bronchial Epithelial Cell Model

Background

In response to viral infection, bronchial epithelial cells increase inflammatory cytokine release to activate the immune response and curtail viral replication. In atopic asthma, enhanced expression of Th2 cytokines is observed and we postulated that Th2 cytokines may augment the effects of rhinovirus-induced inflammation.

Methods

Primary bronchial epithelial cell cultures from pediatric subjects were treated with Th2 cytokines for 24 h before infection with RV16. Release of IL-8, IP-10 and GM-CSF was measured by ELISA. Infection was quantified using RTqPCR and TCID₅₀. Phosphatidyl inositol 3-kinase (PI3K) and P38 mitogen activated protein kinase (MAPK) inhibitors and dexamethasone were used to investigate differences in signaling pathways.

Results

The presence of Th2 cytokines did not affect RV replication or viral titre, yet there was a synergistic increase in IP-10 release from virally infected cells in the presence of Th2 cytokines. Release of IL-8 and GM-CSF was also augmented. IP-10 release was blocked by a PI3K inhibitor and IL-8 by dexamethasone.

Conclusion

Th2 cytokines increase release of inflammatory cytokines in the presence of rhinovirus infection. This increase is independent of effects of virus replication. Inhibition of the PI3K pathway inhibits IP-10 expression.     

Homocysteinylated albumin promotes increased monocyte-endothelial cell adhesion and up-regulation of MCP1, Hsp60 and ADAM17

Rationale

The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties.

Objective

Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels.

Methods and Results

Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2.

Conclusions

Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.     

Shedding of Collagen XVII/BP180 in Skin Depends on Both ADAM10 and ADAM9

Collagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10^−/− cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9^−/− keratinocytes, and (d) H₂O₂ enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9^−/− mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin.Collagen XVII (also called BP180 or BPAG2) is a hemidesmosomal adhesion component in the skin and mucosa and belongs to the emerging group of collagenous transmembrane proteins (1). This type II oriented transmembrane protein is involved in the molecular pathology of human skin diseases. Mutations in the COL17A1 gene are associated with junctional epidermolysis bullosa, a genetic skin blistering disease (2). Patients with bullous pemphigoid and related autoimmune bullous dermatoses have tissue-bound and circulating autoantibodies targeting collagen XVII (3). Structural and functional changes of collagen XVII play an important role in these diseases, although the molecular pathology is not yet fully understood. The collagen XVII consists of three 180-kDa α1 (XVII) chains, each with an intracellular N-terminal domain, a short transmembrane stretch, and a flexible extracellular C-terminal ectodomain with collagenous (Col)² subdomains that are interrupted by short non-collagenous (NC) sequences. The human and murine collagen XVII molecules differ in size and in the number of the Col and NC domains. Human collagen XVII consists of 1497 amino acid residues with 15 Col and 16 NC domains, whereas the murine form, which is 86% identical (4), consists of 1433 amino acid residues with 13 Col and 14 NC domains. In humans the extracellular linker domain NC16A between the plasma membrane and the Col15 domain is functionally important because it is believed to play a role in both ectodomain shedding and in the proper folding of the triple helical structure of collagen XVII (5–7).Our previous studies revealed two forms of collagen XVII, the 180-kDa membrane-anchored form and the soluble 120-kDa form. The latter represents the extracellular collagenous ectodomain, which is released by cleavage by membrane-anchored metalloproteinases of the a disintegrin and metalloproteinase (ADAM) family (8). The shed ectodomain of collagen XVII is very stable in vivo and in vitro. In wound scratch assays, both addition of the purified soluble ectodomain or overexpression of ADAMs suppressed cell motility (8), indicating that the ectodomain has a role in regulating keratinocyte-matrix interactions. In the context of the known functions of collagen XVII as an adhesion molecule, its shedding could therefore regulate its functions in keratinocyte migration, differentiation, and proliferation.ADAMs are also involved in the release of several other type I or type II transmembrane proteins and are considered to be critical regulators of epidermal growth factor receptor signaling, tumor necrosis factor α release, and Notch signaling to name a few examples (9, 10). Previously ADAM9, ADAM10, and ADAM17 had been identified as potential sheddases for collagen XVII in keratinocytes by overexpression in cell-based assays (8). Moreover Adam17^−/− keratinocytes had 50% diminished collagen XVII shedding, which was interpreted to suggest that ADAM17 represents an important, if not the major, physiological collagen XVII sheddase (8). The major goal of the current study was to further explore the contribution of ADAM17 and other candidate sheddases to the release of collagen XVII from primary keratinocytes and mouse skin. The identification of the major collagen XVII sheddases and their regulation is critical for understanding the role of collagen XVII shedding in the pathogenesis of skin diseases.     

ADAM10 Releases a Soluble Form of the GPNMB/Osteoactivin Extracellular Domain with Angiogenic Properties

Background

Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein expressed in approximately 40–75% of breast cancers. GPNMB/OA promotes the migration, invasion and metastasis of breast cancer cells; it is commonly expressed in basal/triple-negative breast tumors and is associated with shorter recurrence-free and overall survival times in patients with breast cancer. Thus, GPNMB/OA represents an attractive target for therapeutic intervention in breast cancer; however, little is known about the functions of GPNMB/OA within the primary tumor microenvironment.

Methodology/Principal Findings

We have employed mouse and human breast cancer cells to investigate the effects of GPNMB/OA on tumor growth and angiogenesis. GPNMB/OA-expressing tumors display elevated endothelial recruitment and reduced apoptosis when compared to vector control-derived tumors. Primary human breast cancers characterized by high vascular density also display elevated levels of GPNMB/OA when compared to those with low vascular density. Using immunoblot and ELISA assays, we demonstrate the GPNMB/OA ectodomain is shed from the surface of breast cancer cells. Transient siRNA-mediated knockdown studies of known sheddases identified ADAM10 as the protease responsible for GPNMB/OA processing. Finally, we demonstrate that the shed extracellular domain (ECD) of GPNMB/OA can promote endothelial migration in vitro.

Conclusions/Significance

GPNMB/OA expression promotes tumor growth, which is associated with enhanced endothelial recruitment. We identify ADAM10 as a sheddase capable of releasing the GPNMB/OA ectodomain from the surface of breast cancer cells, which induces endothelial cell migration. Thus, ectodomain shedding may serve as a novel mechanism by which GPNMB/OA promotes angiogenesis in breast cancer.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

IL-10 Modulates In Vitro Multinucleate Giant Cell Formation in Human Tuberculosis

Background

Multinucleated giant cells (MGC) are the histologic hallmark of granuloma which is known to limit tuberculosis infection. Both Th1 and Th2 type of cytokines regulate the immune response occurring within the granulomas. The objective of the study was to determine whether tuberculosis patient monocytes differed in their MGC forming ability as compared to healthy controls.

Methods

In vitro MGC formation was carried out by treatment of monocytes with cytokine containing culture supernatant of ConA or PPD stimulated peripheral mononuclear cells. IL-2, TNF-α, IL-4, IL-10 and TGF-β cytokine levels were analysed in culture supernatants using ELISA. IL-4 and IL-10 were added to culture supernatant separately and simultaneously along with their respective neutralizing antibodies and their consequent effect on MGC formation was evaluated.

Results

MGC formation was significantly low in patient monocytes incubated with autologous culture supernatant as compared to control culture supernatant. Cytokine analysis of the culture supernatants revealed that while IL-4 levels were similar in patients and controls, increased IL-10 levels were found in patients. Exogenous addition of IL-10 resulted in reduced MGC formation. Contrastingly, when IL-4 was added exogenously, it led to increased MGC formation. The effects of both IL-10 and IL-4 were reversed upon addition of their respective antibodies.

Conclusion

The findings suggest that one of the factors contributing to the disease could be the effect of cytokines on the functionality of monocytes, which are crucial in the fight against the organism. Significantly reduced MGC formation was observed on addition of IL-10. The findings imply an overriding role of IL-10 in MGC formation. The suppressive effect of IL-10 on MGC formation was further confirmed by addition of IL-10 neutralizing antibody.