A Disintegrin And Metalloproteinase Research Articles

ADAM28: Another ambivalent protease in cancer.

Emergence of novel therapeutic options in a perspective of personalized therapy of cancer relies on the discovery of precise molecular mechanisms involved in the switch from a localized tumor to invasive metastasis spread. Pro-tumor functions have been mostly ascribed to proteolytic enzymes from the metalloproteinase family including A Disintegrin And Metalloproteinases (ADAMs). Particularly, when expressed by cancer cells, ADAM28 protease supports cancer cell proliferation, survival and migration as well as metastatic progression. In sharp contrast, ADAM28 derived from the tumor microenvironment has shown to exert strong protective effects against deleterious metastasis dissemination. Indeed, depletion of host-derived ADAM28 (ADAM28 KO mice) accelerates colonization lung tissues, increases tumor foci implantation, and impairs T cell immune response. In this review, we outline specific ADAM28 functions when specifically expressed by carcinoma cells or by tumor microenvironment. Finally, we discuss about future research strategies that could be pursued to highlight new functions of this protease in cancer.

Prognostic Significance of ADAM17 for Gastric Cancer Survival: A Meta-Analysis.

Background and objectives: The prognostic role of a disintegrin and metalloproteinase (ADAM) 17 has been widely assessed in gastric cancer. However, the results are inconsistent. We performed a meta-analysis to evaluate the prognostic significance of ADAM17 and its association with clinicopathological parameters. Methods: The databases of PubMed, Web of Science, and Embase were searched for relevant articles published up to April 2020. The reported hazard ratios (HRs) and odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were pooled to evaluate the strength of the association. Stata 12.1 was used to perform statistical analyses. Results: Seven studies, including 1757 patients, were screened for the meta-analysis. Compared with the high ADAM17 expression group, the pooled HR was higher in the low ADAM17 expression group (HR = 2.04, 95% CI 1.66–2.50; I2 = 18.1%; p = 0.299). High ADAM17 expression was also related to the tumor node metastasis (TNM) stages (OR = 4.09, 95% CI 1.85–9.04; I2 = 84.1%; p = 0.000), lymph node metastasis (OR = 3.08, 95% CI 1.13–8.36; I2 = 79.7%; p = 0.007), and ages (OR = 1.65, 95% CI 1.24–2.21; I2 = 0%; p = 0.692) of the gastric patients. Conclusions: This meta-analysis revealed that ADAM17 is a significant biomarker for poor prognosis in gastric cancer.

ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation

A disintegrin and metalloproteinase (ADAM)12 was previously found to be expressed in T cells in the inflamed brain. However, the function of ADAM12 in T-cell responses in general and in tissue inflammation has not been examined. Here, we studied the role of ADAM12 in T-cell responses, fate determination on activation, and its functions in T cells to mediate tissue inflammation. We identified ADAM12 as a costimulatory molecule that is expressed on naive T cells and downregulated on stimulation. ADAM12 mimics CD28 costimulatory signaling to activate and induce the proliferation of T-helper 1 (Th1) cells. Monoclonal ADAM12 Fab antibodies trigger T-cell activation by amplifying TCR signaling to stimulate T-bet-mediated IFNγ production. Lack of genomic ADAM12 and its knockdown in T cells diminished T-bet and IFNγ production in Th1 cells, whereas other T cells, including Th17 cells, were unaffected. ADAM12 had similar functions in vivo on myelin antigen (MOG35–55)-induced T-cell activation. We found that genetic loss of ADAM12 profoundly alleviated Th1-mediated neuroinflammation and thus disease severity in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Transcriptomic profiling of MOG35–55-specific ADAM12−/− T cells revealed differentially expressed genes that are important for T-cell activation, proliferation, and costimulatory signaling and Th1 pathogenicity, consistent with their inability to cause T-cell-mediated skin inflammation in a model of adoptive delayed-type hypersensitivity. We conclude that ADAM12 is a T-cell costimulatory molecule that contributes to the pathogenesis of tissue inflammation and a potential target for the treatment of Th1-mediated diseases.

Biology of Tissue Inhibitor of Metalloproteinase 3 (TIMP3), and Its Therapeutic Implications in Cardiovascular Pathology.

Tissue inhibitor of metalloproteinase 3 (TIMP3) is unique among the four TIMPs due to its extracellular matrix (ECM)-binding property and broad range of inhibitory substrates that includes matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and ADAM with thrombospondin motifs (ADAMTSs). In addition to its metalloproteinase-inhibitory function, TIMP3 can interact with proteins in the extracellular space resulting in its multifarious functions. TIMP3 mRNA has a long 3’ untranslated region (UTR) which is a target for numerous microRNAs. TIMP3 levels are reduced in various cardiovascular diseases, and studies have shown that TIMP3 replenishment ameliorates the disease, suggesting a therapeutic potential for TIMP3 in cardiovascular diseases. While significant efforts have been made in identifying the effector targets of TIMP3, the regulatory mechanism for the expression of this multi-functional TIMP has been less explored. Here, we provide an overview of TIMP3 gene structure, transcriptional and post-transcriptional regulators (transcription factors and microRNAs), protein structure and partners, its role in cardiovascular pathology and its application as therapy, while also drawing reference from TIMP3 function in other diseases.

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

Two forms of CX3CL1 display differential activity and rescue cognitive deficits in CX3CL1 knockout mice

BackgroundFractalkine (CX3CL1; FKN) is a chemokine expressed by neurons that mediates communication between neurons and microglia. By regulating microglial activity, CX3CL1 can mitigate the damaging effects of chronic microglial inflammation within the brain, a state that plays a major role in aging and neurodegeneration. CX3CL1 is present in two forms, a full-length membrane-bound form and a soluble cleaved form (sFKN), generated by a disintegrin and metalloproteinase (ADAM) 10 or 17. Levels of sFKN decrease with aging, which could lead to enhanced inflammation, deficits in synaptic remodeling, and subsequent declines in cognition. Recently, the idea that these two forms of CX3CL1 may display differential activities within the CNS has garnered increased attention, but remains unresolved.MethodsHere, we assessed the consequences of CX3CL1 knockout (CX3CL1-/-) on cognitive behavior as well as the functional rescue with the two different forms of CX3CL1 in mice. CX3CL1-/- mice were treated with adeno-associated virus (AAV) expressing either green fluorescent protein (GFP), sFKN, or an obligate membrane-bound form of CX3CL1 (mFKN) and then subjected to behavioral testing to assess cognition and motor function. Following behavioral analysis, brains were collected and analyzed for markers of neurogenesis, or prepared for electrophysiology to measure long-term potentiation (LTP) in hippocampal slices.ResultsCX3CL1−/− mice showed significant deficits in cognitive tasks for long-term memory and spatial learning and memory in addition to demonstrating enhanced basal motor performance. These alterations correlated with deficits in both hippocampal neurogenesis and LTP. Treatment of CX3CL1−/− mice with AAV-sFKN partially corrected changes in both cognitive and motor function and restored neurogenesis and LTP to levels similar to wild-type animals. Treatment with AAV-mFKN partially restored spatial learning and memory in CX3CL1−/− mice, but did not rescue long-term memory, or neurogenesis.ConclusionsThese results are the first to demonstrate that CX3CL1 knockout causes significant cognitive deficits that can be rescued by treatment with sFKN and only partially rescued with mFKN. This suggests that treatments that restore signaling of soluble forms of CX3CL1 may be a viable therapeutic option for aging and disease.

Macrolets: Outsized Extracellular Vesicles Released from Lipopolysaccharide-Stimulated Macrophages that Trap and Kill Escherichia coli.

SummaryMacrophages release a variety of extracellular vesicles (EVs). Here we describe a previously unreported class of EVs that are released from macrophages in response to Escherichia coli endotoxin, lipopolysaccharide (LPS), that we have named "macrolets" since they are extruded as large "droplets" released from macrophages. Morphologically, macrolets are anuclear, bounded by a single lipid membrane and structurally dependent on an actin cytoskeleton. Macrolets are enriched in tetraspanins and separable on this basis from their parent macrophages. Macrolets are distinguished from classic exosomes by their larger size (10–30 μm), discoid shape, and the presence of organelles. Macrolets are rich in both interleukin 6 (IL-6) and interleukin 6 receptor (IL-6R),and are capable of trapping and killing E. coli in association with production of reactive oxygen species. Our observations offer insights into the mechanisms by which macrophage activities may be amplified in sites of infection, inflammation, and healing.

The Role of a Disintegrin and Metalloproteinase Proteolysis and Mechanical Damage in Nonphysiological Shear Stress-Induced Platelet Receptor Shedding.

In order to explore the role of a disintegrin and metalloproteinase (ADAM) proteolysis and direct mechanical damage in non-physiologic shear stress (NPSS)-caused platelet receptor shedding, the healthy donor blood treated with/without ADAM inhibitor was exposed to NPSS (150 Pa). The expression of the platelet surface receptors glycoprotein (GP) Ibα and glycoprotein (GP) VI (GPVI) in NPSS-damaged blood was quantified with flow cytometry. The impact of ADAM inhibition on adhesion of NPSS-damaged platelets on von Willibrand factor (VWF) and collagen was explored with fluorescence microscopy. The impact of ADAM inhibition on ristocetin- and collagen-caused aggregation of NPSS-damaged platelets was examined by aggregometry. The results showed that ADAM inhibition could lessen the NPSS-induced loss of platelet surface receptor GPIbα (12%) and GPVI (9%), moderately preserve adhesion of platelets on VWF (7.4%) and collagen (8.4%), and partially restore the aggregation of NPSS-sheared platelets induced by ristocetin (18.6 AU*min) and collagen (48.2 AU*min). These results indicated that ADAM proteolysis played a role in NPSS-induced receptor shedding. However, the ADAM inhibition couldn't completely suppress the NPSS-caused loss of the platelet surface receptors (GPIbα and GPVI), only partially prevented the NPSS-induced reduction of platelet adhesion to VWF and collagen, and the agonist (ristocetin and collagen)-caused platelet aggregation. These results suggested that the direct mechanical damage is partially responsible for NPSS-induced receptor shedding in addition to the ADAM proteolysis. In conclusion, NPSS relevant to blood contacting medical devices can induce ADAM proteolysis and direct mechanical damage on the platelet receptor GPIbα and GPVI, leading to comprised hemostasis.

Inhibition of Metalloproteinase Activity Promotes PMVEC Barrier Function Under Septic Conditions

BackgroundSepsis is a life‐threatening human disease with significant mortality characterized by excessive inflammation, which can lead to endothelial dysfunction, microvascular injury and organ damage. During sepsis, endothelial cells, especially the pulmonary microvascular endothelial cells (PMVEC), become injured leading to loss of barrier function and accumulation of protein‐rich edematous fluid. Metalloproteinases, including the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) families, are capable of cleaving cell surface proteins, such as cell‐cell junctional proteins, suggesting a potential role in septic PMVEC barrier dysfunction. Metalloproteinase activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). Recent work in our lab found that PMVEC lacking TIMP3, a critical regulator of both MMP and ADAM activity, had increased permeability compared to wild type (WT) PMVEC.HypothesisWe hypothesize that PMVEC‐derived metalloproteinase activity will be increased under septic conditions and that vascular permeability will be reduced with the application of synthetic metalloproteinase inhibitors.Materials and MethodsPMVEC isolated from WT and Timp3−/− mice were stimulated with PBS (control) or cytomix (equimolar tumour necrosis factor α, interferon γ, and interleukin 1β) + lipopolysaccharide (LPS; septic) for 4 hours. Metalloproteinase activity was then assessed in the conditioned media and cell lysate, and trans‐PMVEC macromolecular flux was assessed using Evans blue‐labelled albumin. Additionally, PMVEC surface localization of VE‐cadherin (adherens junction) and claudin‐5 (tight junction) was assessed by immunofluorescence on WT and Timp3−/− PMVEC. To confirm the role of metalloproteinases, WT and Timp3−/− PMVEC were treated with synthetic metalloproteinase inhibitors.ResultsAnalysis of metalloproteinase activity revealed increased MMP13 and ADAM17 activity in septic PMVEC and Timp3−/− PMVEC leading to a loss of inter‐PMVEC junctional proteins and subsequent PMVEC barrier dysfunction. Importantly, the application of synthetic metalloproteinase inhibitors such as BB94, CL82198 and TAPI2 reduced the permeability and disruption of VE‐cadherin and claudin 5 under septic conditions.Discussion and conclusionIncreased metalloproteinase activity under septic conditions is associated with disrupted inter‐PMVEC junctional protein localization and loss of barrier function suggesting metalloproteinases are critical mediators of septic PMVEC barrier dysfunction. Further, PMVEC‐derived TIMP3 appears to be an important regulator of PMVEC‐derived metalloproteinase activity as loss of TIMP3 is associated with increased metalloproteinase activity and subsequent PMVEC barrier dysfunction. Thus, these studies suggest that inhibition of metalloproteinase activity may promote PMVEC barrier function by reducing inter‐junctional protein degradation during sepsis and thereby reducing septic vascular permeability.Support or Funding InformationOTSHeart and Stroke OGS

ADAM17 protects against elastase-induced emphysema by suppressing CD62L+ leukocyte infiltration in mice.

Pulmonary emphysema is a major manifestation of chronic obstructive pulmonary disease and is associated with chronic pulmonary inflammation caused by cigarette smoking, with contributions from immune cells such as neutrophils, macrophages, and lymphocytes. Although matrix metalloproteinases are well known to contribute to emphysema progression, the role of a disintegrin and metalloproteinase (ADAM) family proteins, other major metalloproteinases, in disease pathogenesis is largely unknown. ADAM17 is a major sheddase that cleaves various cell surface proteins, including CD62L, an adhesion molecule that plays a critical role in promoting the migration of immune cells to the site of inflammation. In the present study, we aimed to investigate the potential role of ADAM17 and CD62L in the development of elastase-induced emphysema. Control and Adam17flox/flox/Mx1-Cre (Adam17ΔMx1) mice (8-10 wk old) were intratracheally injected with 5 units of porcine pancreas elastase and monitored for 35 days after injection. Lung alveolar destruction was evaluated by analyzing the mean linear intercepts of lung tissue specimens and by histopathological examination. Mean linear intercepts data indicated that the degree of elastase-induced emphysema was significantly more severe in Adam17ΔMx1 mice. Furthermore, flow cytometry showed that CD62L+ neutrophil, CD62L+ macrophage, and CD62L+ B lymphocyte numbers were significantly increased in Adam17ΔMx1 mice. Moreover, the pharmacological depletion of CD62L+ cells with a CD62L-neutralizing antibody ameliorated the extent of emphysema in Adam17ΔMx1 mice. Collectively, these results suggest that ADAM17 possibly suppresses the progression of emphysema by proteolytically processing CD62L in immune cells and that ADAM17 and CD62L could be novel therapeutic targets for treating pulmonary emphysema.

Substrate-Specific Activation of α-Secretase by 7-Deoxy-Trans-Dihydronarciclasine Increases Non-Amyloidogenic Processing of β-Amyloid Protein Precursor.

Accumulation of β-amyloid (Aβ) in the brain has been implicated in the pathology of Alzheimer’s disease (AD). Aβ is produced from the Aβ precursor protein (APP) through the amyloidogenic pathway by β-, and γ-secretase. Alternatively, APP can be cleaved by α-, and γ-secretase, precluding the production of Aβ. Thus, stimulating α-secretase mediated APP processing is considered a therapeutic option not only for decreasing Aβ production but for increasing neuroprotective sAPPα. We have previously reported that 7-deoxy-trans-dihydronarciclasine (E144), the active component of Lycoris chejuensis, decreases Aβ production by attenuating APP level, and retarding APP maturation. It can also improve cognitive function in the AD model mouse. In this study, we further analyzed the activating effect of E144 on α-secretase. Treatment of E144 increased sAPPα, but decreased β-secretase products from HeLa cells stably transfected with APP. E144 directly activated ADAM10 and ADAM17 in a substrate-specific manner both in cell-based and in cell-free assays. The Lineweaver–Burk plot analysis revealed that E144 enhanced the affinities of A Disintegrin and Metalloproteinases (ADAMs) towards the substrate. Consistent with this result, immunoprecipitation analysis showed that interactions of APP with ADAM10 and ADAM17 were increased by E144. Our results indicate that E144 might be a novel agent for AD treatment as a substrate-specific activator of α-secretase.

Tumor Microenvironment-Associated Extracellular Matrix Components Regulate NK Cell Function.

The tumor microenvironment (TME) is composed of multiple infiltrating host cells (e.g., endothelial cells, fibroblasts, lymphocytes, and myeloid cells), extracellular matrix, and various secreted or cell membrane-presented molecules. Group 1 innate lymphoid cells (ILCs), which includes natural killer (NK) cells and ILC1, contribute to protecting the host against cancer and infection. Both subsets are able to quickly produce cytokines such as interferon gamma (IFN-γ), chemokines, and other growth factors in response to activating signals. However, the TME provides many molecules that can prevent the potential effector function of these cells, thereby protecting the tumor. For example, TME-derived tumor growth factor (TGF)-β and associated members of the superfamily downregulate NK cell cytotoxicity, cytokine secretion, metabolism, proliferation, and induce effector NK cells to upregulate ILC1-like characteristics. In concert, a family of carbohydrate-binding proteins called galectins, which can be produced by different cells composing the TME, can downregulate NK cell function. Matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) are also enzymes that can remodel the extracellular matrix and shred receptors from the tumor cell surface, impairing the activation of NK cells and leading to less effective effector functions. Gaining a better understanding of the characteristics of the TME and its associated factors, such as infiltrating cells and extracellular matrix, could lead to tailoring of new personalized immunotherapy approaches. This review provides an overview of our current knowledge on the impact of the TME and extracellular matrix-associated components on differentiation, impairment, and function of NK cells.

Soluble AXL as a marker of disease progression and survival in melanoma.

Receptor tyrosine kinase AXL is a one-pass transmembrane protein upregulated in cancers and associated with lower survival and therapy resistance. AXL can be cleaved by the A Disintegrin and Metalloproteinases (ADAM)10 and ADAM17, yielding a soluble version of the protein. Elevated soluble AXL (sAXL) has been reported to be associated with disease progression in hepatocellular carcinoma, renal cancer, neurofibromatosis type 1 and inflammatory diseases. In the present work, we analyzed sAXL levels in blood from melanoma patients and showed that sAXL increases with disease progression. Additionally, increased sAXL levels were found correlated with shorter two-year survival in stage IV patients treated with ipilimumab. Furthermore, we showed that sAXL levels were related to the percentage of cells expressing AXL in resected melanoma lymph node metastases. This finding was verified in vitro, where sAXL levels in the cell media corresponded to AXL expression in the cells. AXL inhibition using the small-molecular inhibitor BGB324 reduced sAXL levels, while the cellular expression was elevated through increased protein stability. Our findings signify that quantification of sAXL blood levels is a simple and easily assessable method to determine cellular AXL levels and should be further evaluated for its use as a biomarker of disease progression and treatment response.

Necroptosis, ADAM proteases and intestinal (dys)function.

Recently, an unexpected connection between necroptosis and members of the a disintegrin and metalloproteinase (ADAM) protease family has been reported. Necroptosis represents an important cell death routine which helps to protect from viral, bacterial, fungal and parasitic infections, maintains adult T cell homeostasis and contributes to the elimination of potentially defective organisms before parturition. Equally important for organismal homeostasis, ADAM proteases control cellular processes such as development and differentiation, immune responses or tissue regeneration. Notably, necroptosis as well as ADAM proteases have been implicated in the control of inflammatory responses in the intestine. In this review, we therefore provide an overview of the physiology and pathophysiology of necroptosis, ADAM proteases and intestinal (dys)function, discuss the contribution of necroptosis and ADAMs to intestinal (dys)function, and review the current knowledge on the role of ADAMs in necroptotic signaling.

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

A disintegrin and metalloproteinase 12 prevents heart failure by regulating cardiac hypertrophy and fibrosis.

A disintegrin and metalloproteinase (ADAM)12 is considered to promote cardiac dysfunction based on the finding that a small-molecule ADAM12 inhibitor, KB-R7785, ameliorated cardiac function in a transverse aortic constriction (TAC) model by inhibiting the proteolytic activation of heparin-binding-EGF signaling. However, this compound has poor selectivity for ADAM12, and the role of ADAM12 in cardiac dysfunction has not yet been investigated using genetic loss-of-function mice. We revealed that ADAM12 knockout mice showed significantly more advanced cardiac hypertrophy and higher mortality rates than wild-type mice 4 wk after TAC surgery. An ADAM12 deficiency resulted in significantly more expanded cardiac fibrosis accompanied by increased collagen-related gene expression in failing hearts. The results of a genome-wide transcriptional analysis suggested a strongly enhanced focal adhesion- and fibrosis-related signaling pathway in ADAM12 knockout hearts. The loss of ADAM12 increased the abundance of the integrinβ1 subunit and transforming growth factor (TGF)-β receptor types I and III, and this was followed by the phosphorylation of focal adhesion kinase, Akt, mammalian target of rapamycin, ERK, and Smad2/3 in the heart, which resulted in cardiac dysfunction. The present results revealed that the loss of ADAM12 enhanced focal adhesion and canonical TGF-β signaling by regulating the abundance of the integrinβ1 and TGF-β receptors.NEW & NOTEWORTHY In contrast to a long-believed cardio-damaging role of a disintegrin and metalloproteinase (ADAM)12, cardiac hypertrophy was more severe, cardiac function was lower, and mortality was higher in ADAM12 knockout mice than in wild-type mice after transverse aortic constriction surgery. The loss of ADAM12 enhanced focal adhesion- and fibrosis-related signaling pathways in the heart, which may compromise cardiac function. These results provide insights for the development of novel therapeutics that target ADAM12 to treat heart failure.

Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2–processing properties

Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.

Protease Activity in Vascular Disease.

Protease Activity in Vascular Disease.

L1 Cell Adhesion Molecule Overexpression Down Regulates Phosphacan and Up Regulates Structural Plasticity-Related Genes Rostral and Caudal to the Complete Spinal Cord Transection

L1 cell adhesion molecule (L1CAM) supports spinal cord cellular milieu after contusion and compression lesions, contributing to neuroprotection, promoting axonal outgrowth, and reducing outgrowth-inhibitory molecules in lesion proximity. We extended investigations into L1CAM molecular targets and explored long-distance effects of L1CAM rostral and caudal to complete spinal cord transection (SCT) in adult rats. L1CAM overexpression in neurons and glia after Th10/Th11 SCT was achieved using adeno-associated viral vector serotype 5 (AAV5) injected into an L1-lumbar segment immediately after transection. At 5 weeks, a L1CAM mRNA profound decrease detected rostral and caudal to the transection site was alleviated by AAV5-L1CAM treatment, with increased endogenous L1CAM rostral to the SCT. Transected corticospinal tract fibers showed attenuated retraction after treatment, accompanied by a multi-segmental increase of lesion-reduced expression of adenylate cyclase 1 (Adcy1), synaptophysin, growth-associated protein 43, and myelin basic protein genes caudal to transection, and Adcy1 rostral to transection. In parallel, chondroitin sulfate proteoglycan phosphacan elevated after SCT was downregulated after treatment. Low-molecular L1CAM isoforms generated after spinalization indicated the involvement of sheddases in L1CAM processing and long-distance effects. A disintegrin and metalloproteinase (ADAM)10 sheddase immunoreactivity, stronger in AAV5-L1CAM than AAV5- enhanced green fluorescent protein (EGFP)-transduced motoneurons indicated local ADAM10 upregulation by L1CAM. The results suggest that increased L1CAM availability and penetration of diffusible L1CAM fragments post-lesion induce both local and long-distance neuronal and glial responses toward better neuronal maintenance, neurite growth, and myelination. Despite the fact that intervention promoted beneficial molecular changes, kinematic analysis of hindlimb movements showed minor improvement, indicating that spinalized rats require longer L1CAM treatment to regain locomotor functions.

Tspan15 plays a crucial role in metastasis in oral squamous cell carcinoma

Tetraspanin 15 (Tspan15) is a member of the tetraspanin family, which is associated with various biological events and several diseases, however, its role in human oral squamous cell carcinoma (OSCC) remains unknown. The current study aimed to clarify the role of Tspan15 in OSCC. The mRNA and protein expression levels of Tspan15 were up-regulated in OSCC cases and OSCC-derived cell lines. Significant up-regulated Tspan15 expression was found in the advanced OSCC cases; primary tumoral size (P = 0.042), regional lymph node metastasis (P = 0.036) and TNM classification (P = 0.024). The decreased expression of Tspan15 did not significantly affect cellular proliferation, whereas tumoral invasion and migration activities were suppressed in Tspan15-down-regulated cells, suggesting that Tspan15 might activate metastasis-related signaling. Moreover, in the Tspan15-down-regulated cells, the expression of a disintegrin and metalloproteinase (ADAM) 10 was also down-regulated and the cells secreted less soluble N-cadherin compared with control cells. And weak immunoreactivity of β-catenin in the nucleus was detected in Tspan15-down-regulated cells compared with the control cells. These findings suggested that overexpression of Tspan15 positively regulates development of OSCC, and that ADAM10, N-cadherin, β-catenin might be involved in the Tspan15-mediated pathway. These unusual conditions of cell adhesion molecules may lead to high metastasis rate found in Tspan15-overexpressing cases.