Potential role for ADAM15 in pathological neovascularization in mice

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.     

LGI1 and LGI4 bind to ADAM22, ADAM23 and ADAM11

The transmembrane protein ADAM22 is expressed at high levels in the brain. From its molecular structure, ADAM22 is thought to be an adhesion molecule or a receptor because it has functional disintegrin-like and cysteine-rich sequences in its ectodomain. The phenotypic analysis of ADAM22-deficient mice has indicated the important roles played by ADAM22 in proper neuronal function and peripheral nerve development, however, the precise molecular function of ADAM22 is still unknown. To understand the function of ADAM22 on a molecular basis, we identified ADAM22 binding proteins by using immunoprecipitation and mass spectrometric analysis. This analysis revealed that Leucine-rich glioma inactivated 1 (LGI1) is the most potent ADAM22 binding protein in mouse brain. By our quantitative cell-ELISA system, we demonstrated the specific binding of LGI1 with ADAM22. Furthermore, we showed that LGI4, a putative ADAM22 ligand, also bound to ADAM22. Characterization of the binding specificity of LGI1 and LGI4 suggested that ADAM22 is not a sole receptor, because ADAM11 and ADAM23 had a significant binding ability to LGI1 or LGI4. Therefore, LGI-ADAM system seems to be regulated not only by the affinity but also by the cell-type-specific expression of each protein. Our findings provide new clues to understand the functions of LGI1 and LGI4 as an ADAMs ligand.     

SRG4在出生后小鼠睾丸及隐睾中的表达特性

研究小鼠生精新基因SRG4在出生后小鼠睾丸及手术隐睾中的表达特性, 为了解SRG4在精子发生中的作用奠定基础。取出生后1, 3, 12 w小鼠睾丸进行免疫组化检测, 观察SRG4蛋白在出生后小鼠不同发育阶段睾丸中的表达; 制备单侧手术隐睾模型, 取术后0~18 d 的隐睾组织进行半定量RT-PCR检测, 观察SRG4 mRNA在隐睾病变过程中的表达变化, 并对隐睾术后18 d 睾丸进行组织原位杂交分析。免疫组化分析结果表明, SRG4蛋白在出生1 w的小鼠睾丸中几乎检测不到, 在出生3 w的小鼠睾丸中有明显表达, 在出生12 w的小鼠中大量表达, 主要分布在精母细胞和圆形精子细胞胞浆及胞膜, 呈不均匀分布。半定量RT-PCR结果发现, SRG4 mRNA在小鼠隐睾术后0~6 d表达没有明显下调, 9 d 开始表达下调, 第18 d表达最低。组织原位杂交结果表明, 术后18 d隐睾睾丸生殖细胞大量凋亡, 精曲小管中仅见到个别的SRG4阳性信号, 而对照则不受影响。上述结果说明, SRG4蛋白表达受小鼠生长发育调控; 隐睾模型中, 随着生殖细胞的大量凋亡, SRG4基因表达下调, 提示SRG4基因可作为一个精子发生特定阶段的分子标记用以研究精子发生过程。     

ACE2 is expressed in mouse adipocytes and regulated by a high-fat diet

Adipose tissue expresses components of the renin-angiotensin system (RAS). Angiotensin converting enzyme (ACE2), a new component of the RAS, catabolizes the vasoconstrictor peptide ANG II to form the vasodilator angiotensin 1-7 [ANG-(1-7)]. We examined whether adipocytes express ACE2 and its regulation by manipulation of the RAS and by high-fat (HF) feeding. ACE2 mRNA expression increased (threefold) during differentiation of 3T3-L1 adipocytes and was not regulated by manipulation of the RAS. Male C57BL/6 mice were fed low- (LF) or high-fat (HF) diets for 1 wk or 4 mo. At 1 wk of HF feeding, adipose expression of angiotensinogen (twofold) and ACE2 (threefold) increased, but systemic angiotensin peptide concentrations and blood pressure were not altered. At 4 mo of HF feeding, adipose mRNA expression of angiotensinogen (twofold) and ACE2 (threefold) continued to be elevated, and liver angiotensinogen expression increased (twofold). However, adipose tissue from HF mice did not exhibit elevated ACE2 protein or activity. Increased expression of ADAM17, a protease responsible for ACE2 shedding, coincided with reductions in ACE2 activity in 3T3-L1 adipocytes, and an ADAM17 inhibitor decreased media ACE2 activity. Moreover, ADAM17 mRNA expression was increased in adipose tissue from 4-mo HF-fed mice, and plasma ACE2 activity increased. However, HF mice exhibited marked increases in plasma angiotensin peptide concentrations (LF: 2,141 +/- 253; HF: 6,829 +/- 1,075 pg/ml) and elevated blood pressure. These results demonstrate that adipocytes express ACE2 that is dysregulated in HF-fed mice with elevated blood pressure compared with LF controls.     

Biological characterization of ADAM22 variants reveals the importance of a disintegrin domain sequence in cell surface expression

ADAM metallopeptidase domain 22 (ADAM22) is a neuronal membrane-spanning protein that is a potential receptor for leucine-rich, glioma-inactivated 1 (LGI1), and leucine-rich repeat LGI family, member 4 (LGI4). Several lines of study have shown a direct interaction between ADAM22 and LGI1, a mutation which is responsible for inherited epilepsy in humans. Both ADAM22-deficient mice and claw paw mice, congenitally deficient in LGI4, show hypomyelination in the peripheral nerves, suggesting that these molecules are involved in myelination processes. These findings mark ADAM22 as a potential target molecule for epilepsy or demyelination diseases. To investigate the relationship between ADAM22 mutation and its biological character, we designed and examined several ADAM22 variants. We discovered that the ADAM22 P81R variant, the most common polymorphic variation, works as well as the wild-type ADAM22. We also showed that mutations in the disintegrin domain cause a marked decrease in the processing of ADAM22 preproteins, and result in reduced LGI4-binding abilities. Our findings provide valuable information for mutation screening of the ADAM22 gene in patients suffering from epilepsy or demyelinating diseases.     

The Protective Roles of IL‐6 Trans‐Signaling Regulated by ADAM9 on the Liver in Carbon Tetrachloride‐Induced Liver Injury in Mice

Our study was undertaken to evaluate the important role that a disintegrin and metalloproteinase 9 (ADAM9) regulates IL‐6 trans‐signaling in carbon tetrachloride (CCl₄)‐induced liver injury in mice. Mice were divided into four groups. Each group respectively received mineral oil injection, CCl₄ injection, anti‐ADAM9 monoclonal antibody (mAb) pretreatment and CCl₄ injection, anti‐ADAM9 mAb and recombinant mouse ADAM9 molecules pretreatment with CCl₄ injection. Our results showed that anti‐ADAM9 mAb pretreatment significantly aggravated liver injury, inhibited IL‐6 trans‐signaling, which led to downregulation of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), upregulation of Caspase3, cytochrome P450 2E1 (CYP2E1), and hepatocytes apoptosis at 24 h after CCl₄ injection. Recombinant ADAM9 molecules pretreatment reversed the impact of anti‐ADAM9 mAb pretreatment in mice. In conclusion, our study suggested that ADAM9 could regulate the hepatocytes proliferation, apoptosis, angiogenesis, and CYP2E1 expression by activating IL‐6 trans‐signaling and play important protective roles during CCl₄‐induced liver injury in mice.     

miR-29a-3p enhances the radiosensitivity of oral squamous cell carcinoma cells by inhibiting ADAM12

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the head and neck, and radiotherapy is the main approach for this disease, while irradiation resistance is a huge challenge that influences radiosensitivity. This study aims to determine the role and function of miR-29a-3p and ADAM12 in the radiosensitivity of OSCC cells. The expression pattern of ADAM12 in OSCC cells was searched in TCGA database. The binding of miR-29a-3p and ADAM12 was predicted by Starbase and verified using dual luciferase reporter gene assay. The RNA or protein expressions of miR-29a-3p and ADAM12 were measured by RT-qPCR or western blot. OSCC cell lines were treated by various γ-ray irradiation dosages before the alteration on miR-29a-3p expression and on the cell viability, proliferation, migration and cell apoptosis was detected. ADAM12 was highly expressed in OSCC cells, whose expression in resistant cells was positively correlated with irradiation dosage. Overexpression of ADAM12 in OSCC cells lead to increased cell proliferation and migration ability as well as inhibited cell apoptosis. miRNAs potentially binding ADAM12 in PITA, microT, miRmap and targetscan were screened, among which miR-29a-3p had the maximum differential expression levels in OSCC cells determined by RT-qPCR. Overexpression of miR-29a-3p resulted in suppressed cell viability, proliferation, migration ability and increased cell apoptosis, while this expression pattern can be partially counteracted by ADAM12 overexpression in OSCC cells. miR-29a-3p through targeting and inhibiting AMDM12 enhances the radiosensitivity of OSCC cells.Key words: miR-29a, ADAM12, oral squamous cell carcinoma, radio-resistance, cell viability     

Age-dependent pathogenesis of murine gammaherpesvirus 68 infection of the central nervous system

Gammaherpesvirus infection of the central nervous system (CNS) has been linked to various neurological diseases, including meningitis, encephalitis, and multiple sclerosis. However, little is known about the interactions between the virus and the CNS in vitro or in vivo. Murine gammaherpesvirus 68 (MHV-68 or _γHV-68) is genetically related and biologically similar to human gammaherpesviruses, thereby providing a tractable animal model system in which to study both viral pathogenesis and replication. In the present study, we show the successful infection of cultured neuronal cells, microglia, and astrocytes with MHV-68 to various extents. Upon intracerebroventricular injection of a recombinant virus (MHV-68/LacZ) into 4–5-week-old and 9–10-week-old mice, the 4–5-week-old mice displayed high mortality within 5–7 days, while the majority of the 9–10-week-old mice survived until the end of the experimental period. Until a peak at 3–4 days post-infection, viral DNA replication and gene expression were similar in the brains of both mouse groups, but only the 9–10-week-old mice were able to subdue viral DNA replication and gene expression after 5 days post-infection. Pro-inflammatory cytokine mRNAs of tumor necrosis factor-α, interleukin 1β, and interleukin 6 were highly induced in the brains of the 4–5-week-old mice, suggesting their possible contributions as neurotoxic factors in the agedependent control of MHV-68 replication of the CNS. These authors contributed equally to this work.     

Proteomic analysis of protein expression and oxidative modification in r6/2 transgenic mice: a model of Huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.     

The Effect of ADAM8 on the Proliferation and Apoptosis of Hepatocytes and Hepatoma Carcinoma Cells

This study was undertaken to evaluate the effect of ADAM8 on the proliferation and apoptosis of hepatocytes and hepatoma carcinoma cells during hepatocellular carcinoma (HCC) progression. The expression of ADAM8 was significantly increased with good correlation of PCNA expression increasing and cells apoptosis decreasing during the progression of HCC in the liver of mice. Proliferation experiment in vitro showed that recombinant ADAM8 could induce the expression of PCNA in L02 cells, but not in HepG2 cells. Apoptosis experiment in vitro showed that recombinant ADAM8 did not induce or inhibit the expression of apoptosis‐related factors Bcl2, Bax, and Caspase3 in L02 cells, but significantly induced the expression of Bcl2, inhibited the expression of Bax and Caspase3 in HepG2 cells. In conclusion, our study suggested that ADAM8 could promote the proliferation of normal hepatocytes and render hepatoma carcinoma cells more resistant to apoptosis to play important roles during the progression of HCC. ADAM8; Proliferation; Apoptosis     

An ADAM family member with expression in thymic epithelial cells and related tissues

We have analyzed the tissue-specific expression, mRNA isoforms, and genomic structure of murine ADAM28, an ADAM family member recently discovered in human and mouse. While human ADAM28 is expressed in lymphocytes (J. Biol. Chem. 274 (1999) 29251), we observe expression of murine ADAM28 in thymic epithelial cells and developmentally related tissues including the trachea, thyroid, stomach, and lung, but not in lymphocytes. The expression patterns in adult and day 15.5 embryos are similar. We have detected multiple mRNA isoforms varying in the cytoplasmic domain coding sequence and 3prime prime or minute untranslated region due to alternative polyadenylation and splicing events that occur in the final four exons and three introns. The entire ADAM28 gene spans 55 kb and contains 23 exons. The protein sequence contains all conserved residues required for metalloprotease activity, indicative of a role in ectodomain shedding and extracellular matrix modeling. Given its unique expression pattern and potential functions, murine ADAM28 may play a role in organogenesis and organ-specific functions such as thymic T cell development.     

A Disintegrin and Metalloproteinase 17 (ADAM17) Mediates Inflammation-induced Shedding of Syndecan-1 and -4 by Lung Epithelial Cells

Syndecans are cell surface proteoglycans that bind and modulate various proinflammatory mediators and can be proteolytically shed from the cell surface. Within the lung, syndecan-1 and -4 are expressed as transmembrane proteins on epithelial cells and released in the bronchoalveolar fluid during inflammation. We here characterize the mechanism leading to the generation of soluble syndecan-1 and -4 in cultured epithelial cells and murine lung tissue. We show that the bladder carcinoma epithelial cell line ECV304, the lung epithelial cell line A459 and primary alveolar epithelial cells express and constitutively release syndecan-1 and -4. This release involves the activity of the disintegrin-like metalloproteinase ADAM17 as demonstrated by use of specific inhibitors and lentivirally transduced shRNA. Stimulation of epithelial cells with PMA, thrombin, or proinflammatory cytokines (TNFα/IFNγ) led to the down-regulation of surface-expressed syndecan-1 and -4, which was associated with a significant increase of soluble syndecans and cell-associated cleavage fragments. The enhanced syndecan release was not related to gene induction of syndecans or ADAM17, but rather due to increased ADAM17 activity. Soluble syndecan-1 and -4 were also released into the bronchoalveolar fluid of mice. Treatment with TNFα/IFNγ increased ADAM17 activity and syndecan release in murine lungs. Both constitutive and induced syndecan shedding was prevented by the ADAM17 inhibitor. ADAM17 may therefore be an important regulator of syndecan functions on inflamed lung epithelium.     

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane “partner” proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting.     

ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3

Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3. This interaction involved the cysteine-rich domain of ADAM 12. Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12. Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells. Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed. The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.