The role of gicerin, a novel cell adhesion molecule, in development, regeneration and neoplasia

Neurite outgrowth factor (NOF) is an extracellular matrix (ECM) protein in the laminin family and its ligand, gicerin, is a novel cell adhesion molecule in the immunoglobulin superfamily. Gicerin has a homophilic adhesive activity as well as a heterotypic manner to NOF. In the nervous systems, gicerin is expressed during developmental stage when neurons migrate or extend neurites to form a neural network. Gicerin promotes neurite extension and migration of embryonic neurons in vitro by its homophilic and heterophilic adhesion activities. Introduction of antigicerin antibody into early developing eyes perturbs the layer formation of neural retina. These data suggest that gicerin participates in the formation of neural tissues. Gicerin is also expressed in other non-neural tissues; in epithelia of trachea, kidney and oviduct, gicerin expression is restricted in the developmental period. In contrast, muscular tissues and endothelial cells express gicerin continuously even after maturation. Interestingly, gicerin re-appears strongly in the regenerating epithelia of trachea, kidney and oviduct, and also anti-gicerin antibody disrupts the healing process of trachea. Furthermore, gicerin and NOF are overexpressed in the chicken nephroblastomas (Wilm's tumor) and oviductal adenocarcinomas. In vitro analyses show that gicerin adhesive activities can promote binding among tumor cells and adhesion of tumor cells to NOF. A polyclonal antibody against gicerin also perturbs the re-attachment of cancer cells onto metastasizing sites. It is clear from these studies that gicerin is a potential effector for pathological tissue formation as well as for normal development.     

Molecular cloning and analysis of the mouse gicerin gene

Gicerin is a cell adhesion molecule, which has five immunoglobulin-like loop structures in an extracellular domain followed by a single transmembrane domain and a short cytoplasmic tail. We have reported that gicerin participates in neurite extension and structural organization of the nervous system, and its expression in the nervous system is high during the development and dramatically decreased after birth. To elucidate the mechanism how the expression of gicerin is regulated, we performed a genomic cloning of a mouse gicerin. A fragment of 16 kbp genomic clone contained 8 kbp gicerin gene composed of 16 exons with 6 kbp upstream region. Genomic cloning revealed that two isoforms of gicerin were generated by an alternative splicing of exon 15 results in cytoplasmic domains composed of either 63 or 21 amino acids. As for an expressional regulation of gicerin, we found that the mRNA content of gicerin in PC12 cells was regulated by cAMP. Quantitative-PCR analysis revealed that forskolin induced four-fold increase of gicerin mRNA. To characterize the involvement of its promoter region, we examined the promoter activity in PC12 cells by a luciferase-reporter assay. We found that a CRE site located at 60 bp upstream of gicerin gene was responsible for the increase of its mRNA induced by forskolin.     

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.     

Gicerin/CD146 is involved in neurite extension of NGF-treated PC12 cells

Gicerin/CD146 is a cell adhesion molecule, which belongs to the immunoglobulin (Ig) superfamily. We have reported that it has a homophilic binding activity, which participates in the neurite extension from embryonic neurons. To elucidate how gicerin is involved in the neurite extension mechanism, we employed PC12 cells, which expresses gicerin/CD146. PC12 cells extend longer neurites by nerve growth factor (NGF) on gicerin substrate than on without gicerin substrate, which indicates that gicerin participates in neurite extension by NGF. We also found that the expression of gicerin in PC12 cells is induced by NGF. Over-expression of gicerin also promotes neurite extension by gicerin-gicerin homophilic interaction. These findings suggested that increase of gicerin expression by NGF promotes the gicerin-gicerin homophilic interaction resulting in the neurite extension.     

Neuron-specific expression of a chicken gicerin cDNA in transient transgenic zebrafish

Gicerin, a novel cell adhesion molecule which belongs to the immunoglobulin superfamily, is expressed temporally and spatially in the developing chick brain and retina. The previous in vitro experiments using transfected cells showed that gicerin can function as a cell adhesion molecule which has both homophilic and heterophilic binding activities. For the in vivo analyses of gicerin in neural development, we tried to utilize a zebrafish system, a vertebrate suitable for studying early development. We generated transient transgenic animals by microinjecting DNA constructs into zebrafish embryos. Chicken gicerin, under control of the neurofilament gene promoter, was preferentially expressed in neuronal cells and gicerin-expressing neurons exibited a fasciculation formation with neighboring gicerin-positive axons, which may be partly due to homophilic cell adhesion activity of gicerin. These experimental results suggest that this fast and efficient transgenic animal system is useful for studying the functional roles of neuron-specific genes during the development. Special issue dedicated to Dr. Kinya Kuriyama.     

Induction of gicerin/CD146 in the rat carotid artery after balloon injury

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It is reported that the human homologous molecule, CD146, is expressed in the endothelial cells. Here, we found that the expression of gicerin was increased in the rat carotid arteries after balloon injury. Immunohistochemical analysis demonstrated that the expression of gicerin protein was increased in the medial smooth muscle cells prior to the formation of neointima one week after the injury and was also increased in the luminal edge of the neointima after two weeks. We employed A10 cells, a cell line derived from rat aortic smooth muscle cell, and examined the effect of growth factors on the expression of gicerin, such as IGF-1, PDGF-BB, and bFGF. We found that IGF-1, but not PDGF-BB and bFGF, significantly increases the expression of gicerin protein in A10 cells. These suggest gicerin might be involved in the arteriosclerotic neointima formation in the artery.     

Adhesive activity of gicerin, a cell-adhesion molecule, in kidneys and nephroblastomas of chickens

Gicerin, a cell-adhesion molecule belonging to the immunoglobulin superfamily, has both homophilic and heterophilic binding activities to neurite outgrowth factor, an extracellular matrix molecule in the laminin family. Gicerin is thought to play a role in the normal development of chicken kidney, because it is expressed abundantly in the embryonic organ and only slightly in the mature organ. In this study, we have examined the adhesive activity of gicerin in the kidney to characterize its function in organogenesis. We have also examined the function of gicerin in chicken nephroblastomas (“embryonic nephromas”), which show various structures resembling those in embryonic kidneys. Immunohistochemically, the expression patterns of gicerin and neurite outgrowth factor in nephroblastomas are similar to those of embryonic kidneys. Cell-aggregation assays have shown that primary culture cells from both embryonic kidneys and nephroblastomas have strong aggregation activities, and that each aggregation is partially inhibited by gicerin antibody. In contrast, cells from adult kidney exhibit weak aggregation activity that is not inhibited by the antibody. In addition, ligand blot analysis has revealed that gicerins in embryonic kidney and nephroblastoma bind to purified neurite outgrowth factor, whereas extracts from adult kidney show no positive reaction. These findings suggest that the homophilic and heterophilic adhesive activities of gicerin are involved in the formation of both normal kidney and nephroblastoma.     

Gicerin,a cell adhesion molecule,promotes the metastasis of lymphoma cells of the chicken

Gicerin is an immunoglobulin superfamily cell adhesion molecule purified from chicken gizzards. This molecule displays an adhesive interaction with a laminin-like protein as well as with gicerin itself. Gicerin appears in embryonic tissues and plays a role in chick development through its cell adhesive properties. An increase in gicerin expression is found in some sporadic tumors of the chicken. To elucidate the possible role of gicerin in tumor progression in chickens, we introduced gicerin cDNA into an endogenous gicerin negative lymphoma MDCC-MSB1 cell line, and subsequently analyzed them for changes in their metastatic potentials. After intravenous implantation of the gicerin transfectants into chickens, the metastatic potential to the lung, liver and kidney was enhanced compared with parental MDCC-MSB1 cells. Self-aggregation activity was increased in gicerin transfectants. In addition, adhesive and migratory activities of the gicerin transfectants to the gicerin ligands were enhanced in vitro. These findings indicate that gicerin can contribute to the malignancy and metastatic properties of lymphoma.This work was supported in part by a Grant-in-Aid for Scientific Research (No. 13760210), and a grant for Scientific Research on Priority Areas "Cancer" (No. 12215133), from the Ministry of Education, Science, Sports and Culture, Japan, grants from the Uehara Memorial Foundation and Senri Life Science and a Grant-in-Aid for Advanced Scientific Research from Osaka Prefecture University     

Gicerin,a cell adhesion molecule,participates in the histogenesis of retina

Gicerin is a novel cell adhesion molecule that belongs to the immunoglobulin superfamily. Gicerin protein adheres to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family, and also exhibits homophilic adhesion. Heterophilic adhesion of gicerin to NOF is thought to play an active role in neurite outgrowth of developing retinal cells in vitro. In this study, we examined the adhesion activity of gicerin during the retinal development of Japanese quail using an antibody directed against gicerin, to elucidate the biological importance of gicerin in retinal histogenesis. Immunohistochemical and Western blot analysis showed that gicerin was highly expressed in the developing retina but suppressed in the mature retina. The aggregation of neural retinal cells from 5-day embryonic quail retina was significantly inhibited when incubated with a polyclonal antibody to gicerin, suggesting that gicerin protein participates in the adhesion of neural retinal cells of the developing retina. Furthermore, histogenesis of retina both in the organ cultures and in ovo embryos was severely disrupted by incubation with a gicerin antibody. These findings provide evidence that gicerin plays an important role in retinal histogenesis. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 769–780, 1997     

Gicerin,an Ig-superfamily cell adhesion molecule,promotes the invasive and metastatic activities of a mouse fibroblast cell line

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and plays an important role during development through its adhesive properties. Gicerin has two isoforms that differ in their cytoplasmic domains; s-gicerin is the shorter and l-gicerin the longer form of the protein. Gicerin is over-expressed in some sporadic tumors as well as in developing tissues. To provide direct evidence that gicerin has the potential to participate in malignant aspects of tumor cell behavior, a gicerin cDNA was introduced into L-929 cells, an endogenous gicerin-negative mouse fibroblast and subsequently analyzed for changes in their invasive and metastatic potential by implantation into nude mice and chick embryos. Compared with parental cells, both gicerin isoform transfectants showed an enhanced cell growth and invaded deeply into surrounding tissues from implanted sites in both animal models. Furthermore, l-gicerin transfectants markedly enhanced metastasis to the lung. These findings suggest that gicerin promotes the tumor growth and invasion, and the isoform bearing the longer cytoplasmic domain may play a role in metastasis.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.     

Expression of cell adhesion molecule,Gicerin/CD146 during the formation of heart and in the cardiac hypertrophy

Molecular and Cellular Biochemistry - Gicerin/CD146 is a cell adhesion molecule which belongs to the immunoglobulin (Ig) superfamily. We have reported the existence of gicerin/CD146 in the nervous...     

F-spondin and mindin: two structurally and functionally related genes expressed in the hippocampus that promote outgrowth of embryonic hippocampal neurons.

Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.     

Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin-Fc chimeric protein-coated dishes and NOF-coated dishes were carried out. It was found that gicerin-gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti-gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of chick sciatic nerve

We have examined the role of gicerin, an immunoglobulin superfamily cell adhesion molecule, in chick sciatic nerves during development and regeneration. Gicerin was expressed in the spinal cord, dorsal root ganglion (DRG) and sciatic nerves in embryos, but declined after hatching. Neurite extensions from explant cultures of the DRG were promoted on gicerin's ligands, which were inhibited by an anti-gicerin antibody. Furthermore, gicerin expression was upregulated in the regenerating sciatic nerves, DRG and dorsal horn of the spinal cord after injury to the sciatic nerve. These results indicate that gicerin might participate in the development and regeneration of sciatic nerves.     

Expression of neurite outgrowth factor and gicerin during inner ear development and hair cell regeneration in the chick

Several cell adhesion molecules are expressed in the developing inner ear. The present study focused on gicerin, a novel member of the immunoglobulin superfamily, in an attempt to improve our understanding of the development and regeneration of chick inner ear. Gicerin is known to homophilically interact with itself and to bind to neurite outgrowth factor (NOF). The data collected herein show that gicerin is highly expressed in auditory epithelium and acoustic ganglion during early embryogenesis. The immunoreactivity of gicerin in the auditory epithelium decreases more rapidly than that in the acoustic ganglion as the mature hair cells become distinguishable. At the post-hatch stage, the expression of gicerin is not observed. In contrast, NOF was expressed on the basement membranes around the auditory epithelium, and in the acoustic ganglion during development and after birth, but not in the auditory epithelium. Following noise damage, gicerin is transiently re-expressed on the damage receptor epithelium when active cell proliferation is observed in the epithelium. This positive reaction immediately disappears as immature short hair cells appear. These results suggest that gicerin may be associated with cell proliferation in the auditory epithelium, and play a role in neurite extension of the acoustic ganglion cells in conjunction with NOF.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.     

Involvement of Gicerin in the Extension of Microvilli

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. To study the functional differences between l- and s-gicerin, we first examined the distribution of endogenous gicerin in B16 cells and found that l-gicerin was densely localized in microvilli. To clarify the relationship between gicerin and the microvilli, we established independent stable cell lines expressing l- and s-gicerin in L cells and found that l-gicerin localized to the microvilli. Scanning electron microscopic analysis revealed that the microvilli of l-gicerin-transfected cells were longer than those of s-gicerin and control transfectants. This suggested that l-gicerin might participate in the elongation of the microvilli. When cells were double-stained with antibodies to gicerin and moesin, a microvilli-specific protein, the staining of l-gicerin corresponded to that of moesin in the elongated microvilli. Moesin was coprecipitated with glutathione S-transferase-fusion proteins of the l-gicerin cytoplasmic domain but not with the s-gicerin cytoplasmic domain. To determine the region involved in the extension of microvilli, we generated transfectants of two truncated forms of l-gicerin cytoplasmic domain, and we found that only the transfectants of the longer mutant had the longer microvilli, while the shorter mutant exhibited short microvilli. These results suggested that l-gicerin-specific amino acid residues, especially amino acids 16-39, within the cytoplasmic domain of l-gicerin might be involved in the extension of microvilli.     

Identification,characterization, and functional study of the two novel human members of the semaphorin gene family

We cloned two novel human transmembrane semaphorins, (HSA)SEMA6C and (HSA)SEMA6D, that belong to the class VI subgroup of the semaphorin family. The genes for SEMA6C and SEMA6D are mapped on chromosome 1q12-21.1 and 15q21.1, respectively. Among the adult tissues, SEMA6C is expressed only in skeletal muscle, whereas SEMA6D is expressed abundantly in kidney, brain, and placenta and moderately in the heart and skeletal muscles. During murine development, neither SEMA6C nor SEMA6D was expressed in embryonic day 10.5 (E10.5) embryos, but both were highly expressed in the areas of the lateral ventricle, the striatum, the wall of the midbrain, the pons/midbrain junction, and the choroid plexus of E13 embryos. Were neurons, neither axons nor astrocytes, highly expressed both semaphorins. Three isoforms of SEMA6C and five isoforms of SEMA6D derived from alternative splicing were identified, and their expression was regulated in a tissue- and development-dependent manner. Deletion analysis indicated that a sema domain and a PSI domain are integrally necessary for correct post-translation modification and subcellular localization. The extracellular domain of SEMA6C inhibited axonal extension of nerve growth factor-differentiated PC12 cells and induced the growth cone collapse of chicken dorsal root ganglion, rat hippocampal neurons, and rat cortical neurons in a dose-responsive manner. SEMA6D acted like SEMA6C except it had no significant effect on the growth cones of rat cortical neurons.     

Modification of choline acetyltransferase by integration of green fluorescent protein does not affect enzyme activity and subcellular distribution

Choline acetyltransferase (ChAT) is widely used as a marker enzyme to identify cholinergic neurons in the central and peripheral nervous system and to study developmental changes. In order to visualize expression of ChAT directly we have generated a ChAT-green fluorescent protein (GFP) fusion construct. Upon transfection of COS-1 cells and cultured rat hippocampal neurons, transgenic enzymatically active ChAT-GFP is expressed and shows intrinsic fluorescence. In COS-1 cells the ChAT-GFP construct revealed a subcellular distribution indistinguishable from wild-type ChAT. In primary neurons the fluorescence was present in the soma and neuritic processes. Hence, this construct will be useful for analyzing the expression and subcellular distribution of ChAT-GFP in cell and tissue culture.