Regional expression of the ADAMs in developing chicken cochlea

The expression patterns of five members of the ADAM (a disintegrin and metalloprotease) family including ADAM9, ADAM10, ADAM17, ADAM22, and ADAM23 were analyzed in different anatomical structures of the developing chicken cochlea by in situ hybridization and immunohistochemistry. Results show that ADAM9, ADAM10, and ADAM17 are widely expressed in the sensory epithelium of the basilar papilla, by homogene cells, spindle‐shaped cells, and acoustic ganglion cells, and in the tegmentum vasculosum, each with a different pattern. ADAM22 expression is restricted to spindle‐shaped cells and acoustic ganglion cells, while ADAM23 is prominently expressed by hair cells and acoustic ganglion cells. Furthermore, ADAM10 protein is coexpressed with several members of the classic cadherins, including cadherin‐7, N‐cadherin, and R‐cadherin in distinct anatomical regions of the cochlea except for acoustic ganglion cells. The expression of the ADAMs in the developing cochlea suggests a contribution of the ADAMs to the development of distinct cochlear structures. Developmental Dynamics 239:2256–2265, 2010. © 2010 Wiley‐Liss, Inc.     

Differential expression of five members of the ADAM family in the developing chicken brain

ADAMs (a disintegrin and metalloprotease) are a family of trans-membrane multi-domain metalloproteases with multiple functions. So far, more than 35 ADAM family members have been identified from mammalian and nonmammalian sources. Although some functions of ADAMs have been elucidated, their expression patterns remain poorly investigated, especially during CNS development. Here, we cloned the open reading frames or full-length cDNAs of ADAM9, ADAM10, ADAM12, ADAM22 and ADAM23 from chicken embryonic brain, analyzed their evolutionary relationship, and mapped their expression in the embryonic chicken brain by in situ hybridization for the first time. In general, each of the five ADAMs shows a spatially restricted and temporally regulated expression profile. However, the types of tissues and cells, which express each of the five ADAMs, differ from each other. ADAM9 is predominantly expressed in the choroid plexus and in the ventricular layer. ADAM10 is expressed by developing blood vessels, oligodendrocytes, and subsets of neurons and brain nuclei. ADAM12 is expressed by very few brain nuclei, cerebellar Purkinje cells, restricted regions of the neuroepithelium, and some neurons in the deep tectal layers. ADAM22 expression is strong in some brain nuclei and in the pineal gland. ADAM23 is expressed by most gray matter regions and the choroid plexus. The differential expression patterns suggest that the five ADAMs play multiple and versatile roles during brain development.     

Differential regional expression of multiple ADAMs during feather bud formation

The expression of seven members of the ADAM family was investigated by in situ hybridization in the developing feather buds of chicken. The expression profiles of the ADAMs in the cells and tissues of the feather buds differ from each other. ADAM9, ADAM10, and ADAM17 are expressed in the epidermis of the feather bud, whereas ADAM23 expression is restricted to the bud crest, with a distribution similar to that of sonic hedgehog. ADAM13 is not only expressed in the epidermis, but also in restricted regions of the dermis. Both ADAM12 and ADAM22 are expressed in the dermis of the feather bud, with an opposite mediolateral and anteroposterior polarity. Furthermore, the mRNAs of all investigated ADAMs show regional differences in their expression, for example, in the neck and in the roots of the leg and wing. These results suggest that ADAMs play a variety of roles during avian feather bud formation. Developmental Dynamics 240:2142–2152, 2011. © 2011 Wiley‐Liss, Inc.     

Developmental expression of metalloproteases ADAM 9, 10, and 17 becomes restricted to divergent pancreatic compartments.

The A Disintegrin And Metalloprotease (ADAM) family of metalloproteases affects a variety of proteins with important roles in development and disease, including growth factors and adhesion molecules. We have analyzed the expression patterns of ADAMs 9, 10, and 17 during pancreas ontogeny. All ADAMs investigated were expressed in the pancreatic anlagen but invariably became restricted to divergent pancreatic compartments. ADAM9 and 17 became restricted to the insulin-producing beta-cells and all islet cells, respectively. During embryogenesis, ADAM10 was detected predominantly in acinar cells, but in the adult, it was localized to the cell surface membrane of both endocrine and exocrine cells. In addition to ADAM9, a potential prognostic factor for ductal cancers, we describe the expression of ADAM10 and ADAM17 in the pancreatic ductal epithelium. Altogether, the dynamic expression profile of the ADAM proteases described here may reflect a functional divergence of these as mediators of pancreas biology.     

Expression of ADAMs (“a disintegrin and metalloprotease”) in the human lung

In view of the associations of “a disintegrin and metalloprotease” (ADAM) with respiratory diseases, we assessed the expression of various ADAMs in human lung tissue. Lung tissue was obtained from nine individuals who underwent surgery for lung cancer or underwent lung transplantation for emphysema. Also, 16HBE 14o- (human bronchial epithelial) and A549 (alveolar type II epithelium-like) cell lines were used. Immunohistochemistry was performed with antibodies recognizing different ADAM domains. The ADAMs were typically distributed over the bronchial epithelium. ADAM8 and ADAM10 were expressed diffusely in all layers of the epithelium. ADAM9, ADAM17, and ADAM19 were predominantly expressed in the apical part of the epithelium, and ADAM33 was predominantly and strongly expressed in basal epithelial cells. In smooth muscle, ADAM19 and ADAM17 were strongly expressed, as was ADAM33, though this expression was weaker. ADAM33 was strongly expressed in vascular endothelium. All ADAMs were generally expressed in inflammatory cells. The typical distribution of ADAMs in the lung, especially in the epithelium, is interesting and suggests a localized function. As most ADAMs are involved in release of (pro-) inflammatory mediators and growth factors, they may play an important role in the first line of defense and in initiation of repair events in the airways.     

The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts

The metalloprotease ADAM 10 is an important APP alpha-secretase candidate, but in vivo proof of this is lacking. Furthermore, invertebrate models point towards a key role of the ADAM 10 orthologues Kuzbanian and sup-17 in Notch signalling. In the mouse, this function is, however, currently attributed to ADAM 17/TACE, while the role of ADAM 10 remains unknown. We have created ADAM 10-deficient mice. They die at day 9.5 of embryogenesis with multiple defects of the developing central nervous system, somites, and cardiovascular system. In situ hybridization revealed a reduced expression of the Notch target gene hes-5 in the neural tube and an increased expression of the Notch ligand dll-1, supporting an important role for ADAM 10 in Notch signalling in the vertebrates as well. Since the early lethality precluded the establishment of primary neuronal cultures, APPs alpha generation was analyzed in embryonic fibroblasts and found to be preserved in 15 out of 17 independently generated ADAM 10-deficient fibroblast cell lines, albeit at a quantitatively more variable level than in controls, whereas a severe reduction was found in only two cases. The variability was not due to differences in genetic background or to variable expression of the alternative alpha-secretase candidates ADAM 9 and ADAM 17. These results indicate, therefore, either a regulation between ADAMs on the post-translational level or that other, not yet known, proteases are able to compensate for ADAM 10 deficiency. Thus, the observed variability, together with recent reports on tissue-specific expression patterns of ADAMs 9, 10 and 17, points to the existence of tissue-specific 'teams' of different proteases exerting alpha-secretase activity.     

Developmental regulation and neuronal expression of the cellular disintegrin ADAM11 gene in mouse nervous system

ADAM11 is the prototype member of the predominantly CNS-associated clade of the ADAM metalloprotease-disintegrins that has been implicated in neural adhesion and axon guidance. The present study describes the spatiotemporal expression pattern of the ADAM11 gene in adult and developing mouse, and identifies the cells expressing the gene. In the adult CNS, ADAM11 mRNA was present throughout the forebrain, including different cortical fields and diencephalic nuclei. In brainstem, low to moderate expression was detected in certain midbrain nuclei, while several pontine and medullary nuclei showed a very strong signal. High expression was observed in the cerebellar cortex and spinal cord. In addition, ADAM11 was expressed in ganglia of the peripheral nervous system (PNS), retinae, testes, liver, and at lower levels in epidermal and mucosal epithelia, kidney, and salivary gland. The expression was localized to neurons in all examined CNS and PNS subfields. During pre- and perinatal development, ADAM11 was differentially expressed both in the developing PNS and CNS, as well as in heart, kidney, eyes, and brown fat.The present results suggest a widespread involvement of ADAM11 in neuron-neuron or neuron-glial cell interactions during development as well as in the adult nervous system. They provide novel complementary information to recently accumulated data on CNS integrin gene expression and offer useful clues for further studies of the neural functions of ADAMs and integrins.     

Expression of intermediate filaments and desmosomal proteins during differentiation of the human spinal cord

Differentiation of the human spinal cord and involution of its caudal end were investigated in 4-9-week human conceptuses using immunofluorescence and electron microscopy. In the spinal cord, several types of intermediate filament proteins and desmoglein were expressed in parallel: in early stages (4 to 6 weeks), neurofilaments were expressed in low amounts only in the neuroblast processes of the marginal layer. At 6 weeks, differences in staining intensity and distribution patterns of neurofilaments became apparent between lumbar and sacrococcygeal (tail) parts of the spinal cord. Neurofilament expression increased in the mantle and marginal layers of the lumbar spinal cord coinciding with advancing neurogenesis. In contrast, neurofilament expression decreased in the sacrococcygeal spinal cord in association with regression of all tail organs. Regression was characterized by the appearance of large amounts of dead cells and macrophages. Strong vimentin expression was found in neuroepithelial (ependymal) cells and in the radial glia of the spinal cord throughout all stages examined. Coexpression of vimentin and glial fibrillary acidic protein was found only in the radial glia in the earliest developmental stage. Desmoglein was expressed in low amounts around the central canal which was probably associated with the immature junctional complexes that were present between ependymal cells. In conclusion, temporal and spatial distribution patterns of intermediate filament proteins in specific cell populations characterizes differentiation and caudal involution of the human spinal cord.     

Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes

The “A Disintegrin And Metalloproteinases” (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.     

Neuroanatomical localization of myelin basic protein in the late first and early second trimester human foetal spinal cord and brainstem

Summary The temporal and spatial expression of myelin basic protein in the first and second trimester human foetal spinal cord and brainstem from 9 to 20 gestational weeks was determined by immunocytochemistry in sections of cervical, thoracic and lumbosacral levels from 41 human foetal spinal cords and ten brainstems. Myelin basic protein-positive oligodendrocytes were observed peripheral to the ependyma at 9–10 gestational weeks. Oligodendrocytes expressing myelin basic protein were seen at 10–12 gestational weeks in the anterior and lateral funiculi. Myelin basic protein was detected later in the posterior funiculi than in the anterolateral white matter and most spinal cord tracts could not be identified by means of variation in myelin basic protein expression. Mylein basic protein was found in the midline of the brainstem at ten gestational weeks and spread laterally during the second trimester. We conclude that in the human foetal spinal cord, myelin basic protein is present by 10 gestational weeks in the anterolateral cervical spinal cord and midline of the brainstem. It is expressed in a rostral-to-caudal and anterolateral-to-posterior manner in most tracts of the spinal cord. However, an exception to these findings is that the fasciculus gracilis, upon developing into a defined region, had more myelin basic protein-positive cells at the lumbar level than in more rostral regions. Definition of the kinetics of myelin basic protein expression in the normal human foetal spinal cord provides a baseline for study of aberrant myelination and demyelination.     

Characterization of a specific interaction between ADAM23 and cellular prion protein

ADAMs are transmembrane proteins implicated in several biological functions, including cytokine and growth factor shedding, fertilization, muscle and nervous system development. Here, we show for the first time that ADAM23, which is predominantly expressed in the central nervous system, co-localizes with cellular prion protein (PrP^C) at plasma membrane of mouse hippocampal neurons and neuroblastoma cells. Co-immunoprecipitation and pull-down assay showed a physical interaction between ADAM23 and both recombinant and endogenous PrP^C. Glycosylation seems to be not relevant to the observed interaction since both ADAM23 and PrP^C recombinant proteins expressed in bacteria or extracted from eukaryotic cells treated with tunicamycin are still able to bind each other. In vitro binding assays also suggested that the disintegrin domain of ADAM23 is able to interact directly with PrP^C. Taken together, these findings point out PrP^C as a novel molecular partner for ADAM23 in the nervous systems.     

Differential expression of ADAM15 and ADAM17 metalloproteases in the rat brain after severe hypobaric hypoxia and hypoxic preconditioning

The ADAMs (a disintegrin and metalloprotease) are a family of membrane-anchored glycoproteins capable of shedding a multitude of proteins from the cell surface. Although ADAMs are being considered as crucial modulators of physiological and pathophysiological processes, their roles in neuronal death/survival are largely unexplored. In the present study, changes in brain expression of ADAM15 and ADAM17 (TACE) have been quantitatively examined in rats in response to injurious severe hypoxia (SH) and in animals which acquired hypoxic tolerance through preconditioning to mild hypoxia prior SH. SH persistently up-regulated ADAM15 mRNA and protein levels in hippocampus and neocortex but not in thalamus or hypothalamus. This effect was not observed in the preconditioned rats tolerant to SH. In contrast, hippocampal levels of ADAM17 mRNA and neocortical levels of ADAM17 mRNA and protein were largely reduced following SH in non-preconditioned rats. Hypoxic preconditioning prevented down-regulation of the adam17 gene and considerably enhanced ADAM17 protein expression in hippocampus and neocortex in response to SH. The present findings implicate ADAM15 in the processes of neuronal hypoxic injury. On the other hand, these results also provide evidence for a pro-survival neuroprotective role of ADAM17 and its engagement in the process of preconditioning-induced hypoxic tolerance. The analysis of the protein levels of soluble and membrane-bound forms of APP in the neocortex and hippocampus of rats subjected to SH and SH with preconditioning has demonstrated that an increased ADAM17 expression in preconditioned animals 24h after hypoxia corresponded to a higher level of soluble form of APP and a reduction of the membrane bound fraction which reflects the role of ADAM17 in APP shedding.     

Implication of ADAM-8, -9, -10, -12, -15, -17, and ADAMTS-1 in implantational remodeling of a mouse uterus

In the present study, whether the ADAM-8, -9, -10, -12, -15, -17, and ADAMTS-1 proteins might play a role in mouse uterus during periimplantation period was investigated. Immunoblotting analyses demonstrated that all ADAM proteins consistently appeared throughout days 1 to 8 of pregnancy but with a variation depending on the species of ADAM gene, the progression of pregnancy, and the site of the uterus. Immunohistochemical analyses indicated that ADAM proteins were localized in the luminal or glandular epithelial layers with a varying intensity depending on the species of ADAM and the progression of pregnancy. Particularly ADAM-8, -12, and -15, were predominantly located in the implantation site of the uterine tissues, whereas little or no protein was localized in the interimplantation site. Based upon these observations, it is suggested that the ADAMs might play an important role in the remodeling of the mouse uterus during the periimplantation period.     

ADAM10 is a principal 'sheddase' of the low-affinity immunoglobulin E receptor CD23

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.     

Developmental changes in the expression of calbindin and potassium-channel subunits Kv3.1b and Kv3.2 in mouse Renshaw cells

One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14.     

胸髓半横断引起腰髓前角钙网蛋白阳性中间神经元突触联系的变化

为检测大鼠胸髓半横断后后肢运动功能状况和腰髓前角中间神经元的突触联系的变化,并分析其相关关系,本实验通过BBB评分、钙网蛋白(CR)和突触囊泡素(SYN)免疫荧光双标记及相关分析方法进行了研究。结果显示:大鼠胸髓半横断后12 h时,后肢运动功能完全丧失,BBB分值下降,从第3 d开始后肢功能逐渐恢复,BBB分值逐渐增加,至第21 d基本恢复正常;同时观察到腰髓损伤侧12 h时CR免疫反应阳性(CR-IR)神经元周围的SYN-IR降低,第3 d时恢复至正常水平,至第21 d时升高;相关分析显示后肢运动功能BBB评分与CR-IR中间神经元周围的SYN表达水平呈正相关(r=0.45,P<0.05)。上述结果提示大鼠胸髓半横断后,与腰髓CR-IR中间神经元形成的突触联系出现可塑性改变,这种可塑性变化可能是后肢运动功能自发性恢复的形态学基础。