ADAM17 overexpression promotes angiogenesis by increasing blood vessel sprouting and pericyte number during brain microvessel development

The angiogenic process is precisely regulated by different molecular mechanisms, with a balance between stimulatory and inhibitory factors in embryonic development. Transmembrane proteins of the ADAM (a disintegrin and metalloprotease) family play a critical role in embryogenesis and are involved in protein ectodomain shedding, as well as cell-cell and cell-matrix interactions. In the present study, we found that ADAM17 is expressed spatiotemporally in the tectal layers during chicken embryonic development. To investigate the effect of ADAM17 overexpression on angiogenesis, chicken ADAM17 plasmids were transfected into the developing tectum in vivo by electroporation. Results showed that overexpression of ADAM17 induces morphological changes of brain microvessels, such as an increase in diameter, of capillary sprouting from radial microvessels and an increase in the number of pericytes, but not of endothelial cells. Our data suggest that overexpression of ADAM17 in the developing tectum promotes angiogenesis by increasing the number of pericytes and capillary sprouting in the radial vessels.     

Catalytic properties of ADAM19

ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.     

Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development

The ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.     

Differential expression of the ADAMs in developing chicken retina

The expression patterns of the seven members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 were analyzed in the developing chicken retina by in situ hybridization and immunohistochemistry. Results show that each individual ADAM is expressed and regulated spatiotemporally in the developing retinal layers. ADAM9, ADAM10 and ADAM17 are widely expressed in the differential layers of the retina throughout the whole embryonic period, while ADAM12 and ADAM13 are mainly expressed in the ganglion cell layer at a later stage. ADAM22 and ADAM23 are restricted to the inner nuclear layer and the ganglion cell layer at a later stage. Furthermore, ADAM10 protein is co-expressed with the four members of the classic cadherins, N-cadherin, R-cadherin, cadherin-6B and cadherin-7 in distinct retinal layers. Therefore, the differential expression of the investigated ADAMs in the developing retina suggests the contribution of them to the retina development.     

Expression patterns of ADAMs in the developing chicken lens

In the present study the expression patterns of ADAM (a disintegrin and metalloprotease) genes in the chicken developing lens were analyzed. Using in situ hybridization, we found that seven members of the ADAM family including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 are expressed in the developing embryonic lens. From embryonic incubation day (E) 2 to E3, most of the ADAMs investigated here are expressed in the lens placode and lens vesicle. From E5 to E7, all seven ADAMs, but predominantly ADAM9 and ADAM10, are throughly expressed in the central epithelium, as well as in the proliferating lens epithelium and the equatorial lens epithelium. From E9 to E14, expression of ADAM9, ADAM10, and ADAM17 decreases moderately in these regions. ADAM12 and ADAM13 are weakly expressed in the central epithelium and the lens epithelium, and are not detectable from E14 onward. ADAM22 and ADAM23 are expressed in the central epithelium, the lens epithelium and the equatorial lens epithelium at E5 and decrease gradually afterwards in the same regions. At E16, only weak ADAM9, ADAM10 and ADAM17 signals are found in the anterior lens epithelium. The changing spatiotemporal expression of the seven ADAMs suggests a regulatory role for these molecules during chicken lens development.     

PACSIN2 is a regulator of the metalloprotease/disintegrin ADAM13

ADAM13 is a cell surface metalloprotease expressed in cephalic neural crest cells during early Xenopus development. The cytoplasmic domain of ADAM13 contains three potential SH3 (Src homology type 3) binding sites, suggesting that this region may support interactions with intracellular proteins. In this report we describe the identification, by a new strategy, of three proteins that bind the ADAM13 cytoplasmic domain in vitro: X-Src1, X-An4, and X-PACSIN2. We focused our study on X-PACSIN2 protein because it colocalizes with ADAM13 in migrating neural crest cells during embryonic development. Using pull-down experiments we show that X-PACSIN2 binds to ADAM13 in vitro. Using Xenopus XTC cells, we demonstrate that ADAM13 and X-PACSIN2 colocalize to membrane ruffles and cytoplasmic vesicles. We also show that X-PACSIN2 overexpression can rescue developmental alterations induced by overexpression of ADAM13, suggesting that both proteins interact in vivo. Finally, our results suggest that X-PACSIN2 overexpression reduces endogenous ADAM13 function while a truncated X-PACSIN2 (DeltaSH3) increases this activity in cephalic neural crest cells. We propose that X-PACSIN2 may regulate ADAM13 activity by influencing either its subcellular localization or its catalytic activity. In agreement with this model, elimination of the ADAM13 cytoplasmic domain increased developmental alterations attributable to ADAM13 proteolytic activity.     

ADAM19 autolysis is activated by LPS and promotes non-classical secretion of cysteine-rich protein 2

ADAM family proteins are type I transmembrane, zinc-dependent metalloproteases. This family has multiple conserved domains, including a signal peptide, a pro-domain, a metalloprotease domain, a disintegrin (DI) domain, a cysteine-rich (Cys) domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic domain. The Cys and DI domains may play active roles in regulating proteolytic activity or substrate specificity. ADAM19 has an autolytic processing activity within its Cys domain, and the processing is necessary for its proteolytic activity. To identify a new physiological function of ADAM19, we screened for associating proteins by using the extracellular domain of ADAM19 in a yeast two-hybrid system. Cysteine-rich protein 2 (CRIP2) showed an association with ADAM19 through its DI and Cys domains. Sequence analysis revealed that CRIP2 is a secretable protein without a classical signal. CRIP2 secretion was increased by overexpression of ADAM19 and decreased by suppression of ADAM19 expression. Moreover, CRIP2 secretion increased in parallel with the autolytic processing of ADAM19 stimulated by lipopolysaccharide. These findings suggest that ADAM19 autolysis is activated by lipopolysaccharide and that ADAM19 promotes the secretion of CRIP2.     

ADAM23 knockdown promotes neuronal differentiation of P19 embryonal carcinoma cells by up‐regulating P27KIP1 expression

ADAM23 (a disintegrin and metalloprotease 23), a member of brain MDC (macrophage‐derived chemokine) family, is important for the development of CNS (central nervous system). P19 mouse embryonal carcinoma cells can differentiate into neurons when cultured in aggregates and induced with RA (retinoic acid). We have found that under conditions without RA induction, knocking down ADAM23 with RNAi (RNA interference) promoted neuronal differentiation, and similarly recombinant GST (glutathione transferase)‐ADAM23‐DIS protein inhibited neuronal differentiation of P19/ADAM23KD (P19/ADAM23‐knockdown) cells. In P19/ADAM23KD, there were more cells arrested in G1 phase than normal P19 cells, due to the up‐regulation of P57^KIP2 and P27^KIP1 expression. P27^KIP1 was up‐regulated during the differentiation process of both P19/ADAM23KD cells without RA induction, and P19 cells with RA induction. Transient overexpression of P27^KIP1 in P19 cells also promoted neuronal differentiation of P19 cells. The findings indicate that ADAM23 suppresses neuronal differentiation through its disintegrin domain, and Adam23 KD up‐regulates P27^KIP1 in P19/ADAM23KD cells, one reason that P19/ADAM23KD cells can differentiate into neurons without RA induction.     

Evaluation of the contribution of different ADAMs to tumor necrosis factor alpha (TNFalpha) shedding and of the function of the TNFalpha ectodomain in ensuring selective stimulated shedding by the TNFalpha convertase (TACE/ADAM17)

Tumor necrosis factor-alpha (TNFalpha), a potent pro-inflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-alpha converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNFalpha in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNFalpha cleavage site in vitro, these enzymes are considered to be candidate TNFalpha sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNFalpha shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester- and pervanadate-stimulated release of TNFalpha in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNFalpha, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNFalpha shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNFalpha to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNFalpha is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNFalpha makes an unexpected contribution to the selective cleavage of TNFalpha by ADAM17: it prevents one or more other enzymes from cleaving TNFalpha following PMA stimulation. Thus, selective stimulated processing of TNFalpha by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site.     

Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse

Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium.     

An actin-depolymerizing protein in embryonic chicken skeletal muscle: purification and characterization

In embryonic skeletal muscle, a large amount of non-polymerized actin exists in the cytoplasm (Shimizu and Obinata [1986] J. Biochem. 99, 751-759). A 19-kDa protein (called 19K protein) which binds to G-actin was purified by sequential chromatography on DNase I-agarose, hydroxylapatite, SP-Sephadex, and Sephadex G-75, from the sarcoplasmic fraction of embryonic chicken skeletal muscle. This protein decreased the extent of actin polymerization at a steady state and increased the monomeric actin in a concentration-dependent fashion; it also caused quick depolymerization of F-actin, as determined by spectrophotometry at 237 nm, viscometry, DNase I inhibition assay, and electron microscopy. The molar ratio of 19K protein and actin interacting with each other was estimated to be 1:1. From these results, 19K protein was regarded as being actin depolymerizing protein. The amount of 19K protein in muscle decreased during development. The inhibitory action of 19K protein was removed by myosin or heavy meromyosin, and actin filaments were formed on the surface of myosin filaments when myosin filaments were added to a mixture of actin and 19K protein in a physiological salt solution. We propose that actin assembly is dually controlled in the developing muscle by the inhibitor(s) and an accelerator (myosin); this mechanism may enable the ordered assembly of actin and myosin in the early phase of myofibrillogenesis.     

A new heparin binding protein regulated by retinoic acid from chick embryo

A 19 KDa heparin binding protein was previously purified from chicken embryos. Essentially localized within basement membranes in early embryonic tissues, this protein is very rich in basic and cystein residues. Its N-terminal fragment is similar to corresponding fragment of two other proteins expressed during embryogenesis and postnatal period. Its synthesis and secretion are induced by retinoic acid in chicken myoblasts and fibroblasts. This new retinoic acid induced heparin binding protein (RI-HB) does stimulate neurite outgrowth and proliferation on PC12 cells. These results suggest that retinoic acid could regulate some aspect of differentiation and development by inducing the synthesis of a new family of growth and neurotrophic factors.     

Evaluation of the contributions of ADAMs 9, 12, 15, 17, and 19 to heart development and ectodomain shedding of neuregulins beta1 and beta2

Defects in heart development are the most common congenital abnormalities in humans, providing a strong incentive to learn more about the underlying causes. Previous studies have implicated the metalloprotease-disintegrins ADAMs (a disintegrin and metalloprotease) 17 and 19 as well as heparin binding EGF-like growth factor (HB-EGF) and neuregulins in heart development in mice. Here, we show that mice lacking both ADAMs 17 and 19 have exacerbated defects in heart development compared to mice lacking either ADAM, providing the first evidence for redundant or compensatory functions of ADAMs in development. Moreover, we identified additional compensatory or redundant roles of ADAMs 9 and 19 in morphogenesis of the mitral valve and cardiac outflow tract. Cell biological studies designed to address the functions of these ADAMs in shedding of HB-EGF uncovered a contribution of ADAM19 to this process, but this was only evident in the absence of the major HB-EGF sheddase, ADAM17. In addition, ADAM17 emerged as the major sheddase for neuregulins beta1 and beta2 in mouse embryonic fibroblasts. These results raise the possibility that ADAMs 9, 17, and 19 contribute to heart development in humans and have implications for understanding the mechanisms underlying congenital heart disease.     

microRNA-33-3p involved in selenium deficiency-induced apoptosis via targeting ADAM10 in the chicken kidney

Selenium (Se) deficiency induces typical clinical and pathological changes and causes various pathological responses at the molecular level in several different chicken organs; the kidney is one of the target organs of Se deficiency. To explore the mechanisms that underlie the effects of microRNA-33-3p (miR-33-3p) on Se deficiency-induced kidney apoptosis, 60 chickens were randomly divided into two groups (30 chickens per group). We found that Se deficiency increased the expression of miR-33-3p in the chicken kidney. A disintegrin and metalloprotease domain 10 (ADAM10) was verified to be a target of miR-33-3p in the chicken kidney. The overexpression of miR-33-3p decreased the expression levels of β-catenin, cyclinD1, T-cell factor (TCF), c-myc, survivin, and Bcl-2; it increased the expression levels of E-cadherin, Bak, Bax, and caspase-3; and it increased the number of chicken kidney cells in the G0/G1 phase. In addition, Se deficiency caused the ultrastructure of the kidney to develop apoptotic characteristics. The results of flow cytometry analysis and AO/EB staining showed that the number of apoptotic chicken kidney cells increased in the miR-33-3p mimic group. All these results suggest that Se deficiency-induced cell cycle arrest and apoptosis in vivo and in vitro in the chicken kidney via the regulation of miR-33-3p, which targets ADAM10.     

ADAM10 Negatively Regulates Neuronal Differentiation during Spinal Cord Development

Members of the ADAM (a disintegrin and metalloprotease) family are involved in embryogenesis and tissue formation via their proteolytic function, cell-cell and cell-matrix interactions. ADAM10 is expressed temporally and spatially in the developing chicken spinal cord, but its function remains elusive. In the present study, we address this question by electroporating ADAM10 specific morpholino antisense oligonucleotides (ADAM10-mo) or dominant-negative ADAM10 (dn-ADAM10) plasmid into the developing chicken spinal cord as well as by in vitro cell culture investigation. Our results show that downregulation of ADAM10 drives precocious differentiation of neural progenitor cells and radial glial cells, resulting in an increase of neurons in the developing spinal cord, even in the prospective ventricular zone. Remarkably, overexpression of the dn-ADAM10 plasmid mutated in the metalloprotease domain (dn-ADAM10-me) mimics the phenotype as found by the ADAM10-mo transfection. Furthermore, in vitro experiments on cultured cells demonstrate that downregulation of ADAM10 decreases the amount of the cleaved intracellular part of Notch1 receptor and its target, and increases the number of βIII-tubulin-positive cells during neural progenitor cell differentiation. Taken together, our data suggest that ADAM10 negatively regulates neuronal differentiation, possibly via its proteolytic effect on the Notch signaling during development of the spinal cord.     

Essential role for ADAM19 in cardiovascular morphogenesis

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19(-/-) animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.