cDNA cloning, chromosomal localization, and expression analysis of human BEHAB/brevican, a brain specific proteoglycan regulated during cortical development and in glioma

BEHAB (Brain Enriched HyAluronan Binding)/brevican, a brain-specific member of the lectican family of chondroitin sulfate proteoglycans (CSPGs), may play a role in both brain development and human glioma. BEHAB/brevican has been cloned from bovine, mouse and rat. Two isoforms have been reported: a full-length isoform that is secreted into the extracellular matrix (ECM) and a shorter isoform with a sequence that predicts a glycophosphatidylinositol (GPI) anchor. Here, we report the characterization of BEHAB/brevican isoforms in human brain. First, BEHAB/brevican maps to human chromosome 1q31. Second, we report the sequence of both isoforms of human BEHAB/brevican. The deduced protein sequence of full-length, secreted human BEHAB/brevican is 89.7, 83.3 and 83.2% identical to bovine, mouse and rat homologues, respectively. Third, by RNase protection analysis (RPA) we show the developmental regulation of BEHAB/brevican isoforms in normal human cortex. The secreted isoform is highly expressed from birth through 8years of age and is downregulated by 20years of age to low levels that are maintained in the normal adult cortex. The GPI isoform is expressed at uniformly low levels throughout development. Fourth, we confirm and extend previous studies from our laboratory, here demonstrating the upregulation of BEHAB/brevican mRNA in human glioma quantitatively. RPA analysis shows that both isoforms are upregulated in glioma, showing an approximately sevenfold increase in expression over normal levels. In contrast to the developmental regulation of BEHAB/brevican, where only the secreted isoform is regulated, both isoforms are increased in parallel in human glioma. The distinct patterns of regulation of expression of the two isoforms suggest distinct mechanisms of regulation of BEHAB/brevican during development and in glioma.     

The proteoglycan brevican binds to fibronectin after proteolytic cleavage and promotes glioma cell motility

The adult neural parenchyma contains a distinctive extracellular matrix that acts as a barrier to cell and neurite motility. Nonneural tumors that metastasize to the central nervous system almost never infiltrate it and instead displace the neural tissue as they grow. In contrast, invasive gliomas disrupt the extracellular matrix and disperse within the neural tissue. A major inhibitory component of the neural matrix is the lectican family of chondroitin sulfate proteoglycans, of which brevican is the most abundant member in the adult brain. Interestingly, brevican is also highly up-regulated in gliomas and promotes glioma dispersion by unknown mechanisms. Here we show that brevican secreted by glioma cells enhances cell adhesion and motility only after proteolytic cleavage. At the molecular level, brevican promotes epidermal growth factor receptor activation, increases the expression of cell adhesion molecules, and promotes the secretion of fibronectin and accumulation of fibronectin microfibrils on the cell surface. Moreover, the N-terminal cleavage product of brevican, but not the full-length protein, associates with fibronectin in cultured cells and in surgical samples of glioma. Taken together, our results provide the first evidence of the cellular and molecular mechanisms that may underlie the motility-promoting role of brevican in primary brain tumors. In addition, these results underscore the important functional implications of brevican processing in glioma progression.     

Reduced Expression of the Hyaluronan and Proteoglycan Link Proteins in Malignant Gliomas

Malignant gliomas have a distinctive ability to infiltrate the brain parenchyma and disrupt the neural extracellular matrix that inhibits motility of axons and normal neural cells. Chondroitin sulfate proteoglycans (CSPGs) are among the major inhibitory components in the neural matrix, but surprisingly, some are up-regulated in gliomas and act as pro-invasive signals. In the normal brain, CSPGs are thought to associate with hyaluronic acid and glycoproteins such as the tenascins and link proteins to form the matrix scaffold. Here, we examined for the first time the expression of link proteins in human brain and malignant gliomas. Our results indicate that HAPLN4 and HAPLN2 are the predominant members of this family in the adult human brain but are strongly reduced in the tumor parenchyma. To test if their absence was related to a pro-invasive gain of function of CSPGs, we expressed HAPLN4 in glioma cells in combination with the CSPG brevican. Surprisingly, HAPLN4 increased glioma cell adhesion and migration and even potentiated the motogenic effect of brevican. Further characterization revealed that HAPLN4 expressed in glioma cells was largely soluble and did not reproduce the strong, hyaluronan-independent association of the native protein to brain subcellular membranes. Taken together, our results suggest that the tumor parenchyma is rich in CSPGs that are not associated to HAPLNs and could instead interact with other extracellular matrix proteins produced by glioma cells. This dissociation may contribute to changes in the matrix scaffold caused by invasive glioma cells.The extracellular matrix (ECM)² of the adult central nervous system lacks most fibrous proteins (collagens, fibronectin, and laminins) that are present in the matrices of other tissues and is formed instead by a scaffold of hyaluronic acid (HA) with associated glycoproteins (1). The major family of HA binding matrix glycoproteins in the central nervous system is formed by the chondroitin sulfate proteoglycans of the lectican family (aggrecan, versican, neurocan, and brevican), the last two expressed almost exclusively in neural tissue (2). These proteoglycans bind both to HA and to cell-surface receptors (3), regulating the cross-linking and compressibility of the matrix scaffold and, therefore, modulating many neural processes including cell motility during development, axonal navigation, and the stabilization of synapses (4). The lecticans have been identified as a major class of molecules that restrict cellular and axonal motility in neural tissue and are a major component of the glial scar that forms after neural injury and prevents axonal regeneration (5).A second family of HA-binding proteins expressed in the central nervous system is formed by small glycoproteins known as HA- and proteoglycan-link proteins (HAPLNs) or, simply, “link proteins.” These glycoproteins bind both to HA and to the lecticans, forming ternary complexes (6, 7). The structure of the link proteins is remarkably similar to the N-terminal region of the lecticans, and the highly homologous HA binding domains from HAPLNs and lecticans are indistinctly known as proteoglycan tandem repeats or link-protein modules.In a striking example of molecular evolution, the genes of the four HAPLNs are located adjacent to the genes of the four lecticans, indicating a common molecular origin by gene duplication (8). Two of the link proteins, HAPLN2 and HAPLN4, have only been detected in neural tissue, and their genes are adjacent to the neural-specific proteoglycans, brevican and neurocan, respectively (8). Both HAPLN2 and HAPLN4, also known as brain-specific link protein (Bral-1) and Bral-2, are up-regulated in the adult central nervous system and match the temporal expression profile of brevican, which is the most abundant CSPG in adult neural tissue (9, 10).Current evidence suggests that the HAPLNs may be key components in the organization of the HA-based matrix scaffold. HAPLN1, the best studied member of the family, increases the affinity of the lecticans for HA (11, 12) and stabilizes lectican-HA matrix aggregates (6, 13). Moreover, the increased expression of lecticans and HAPLNs in the adult central nervous system correlates temporally and spatially with changes in ECM solubility and with appearance of ECM aggregates around subsets of neurons, known as “perineuronal nets.” These changes have been associated with restricted cellular motility and decreased synaptic plasticity (14).The role of the lectican CSPGs as inhibitors of motility in the adult central nervous system contrasts starkly with their pro-invasive role in the highly aggressive brain tumors known as malignant gliomas. These are the most common primary tumors of the brain and are characterized by their extensive and diffuse infiltration of the brain parenchyma (15), which makes them impossible to completely remove and facilitates tumor recurrence even after long term therapies. The invasive ability of gliomas is restricted to neural tissue and is not observed in other tumors that metastasize to the brain, suggesting that glioma invasion may be supported in part by unique mechanisms to remodel the neural microenvironment (16).Two lectican CSPGs, versican and the neural-specific CSPG brevican, are highly up-regulated in gliomas compared with normal brain tissue (3). Although these proteoglycans are thought to inhibit the motility of normal glial cells (17, 18), they instead promote glioma cell adhesion and migration. The underlying molecular mechanisms for this unusual effect are poorly understood, although we and others have demonstrated that these lecticans can activate epidermal growth factor receptor signaling in glioma cells, which leads to an increase of cell-surface adhesion molecules (19). Both brevican and versican can also form adhesive complexes with mesenchymal matrix proteins that are present in the glioma ECM but absent from the normal neural ECM (19, 20).Although the role of CSPGs in brain tumors is starting to become better defined, their HAPLN partners have never been analyzed in human brain or in neuropathologies. Therefore, we still have a highly incomplete picture of the molecular changes that occur in the tumor ECM and of how those changes could affect critical aspects of glioma biology such as invasion of the surrounding tissue.We hypothesized that the gain of function of CSPGs in gliomas could be associated with changes in the levels or molecular associations of specific HAPLNs in the ECM of gliomas. Thus, we studied here the expression and biochemical properties of the HAPLN family in human normal brain and glioma tissue. Our results provide the first biochemical characterization of the brain-specific human HAPLN4 and, in addition, show that both neural-specific link proteins HAPLN2 and HAPLN4, which are abundant in adult brain, are virtually absent from the ECM of malignant gliomas.     

Lack of protein 4.1a in red blood cells of the hereditarily anemic belgrade laboratory (b/b) rat.

We have demonstrated that the red blood cell (RBC) membrane of the hereditarily anemic Belgrade laboratory (b/b) rat contains protein 4. 1b isoform, only. The evidence are given that the synthesis of protein 4.1 in the b/b rat reticulocytes is the same as in normal rat. When haemolytic anaemia was induced in normal rat by in vivo phenyhydrazine treatment the same phenomenon, i.e., the absence of protein 4.1a in the RBC membrane was observed. The increase of 4.1a isoform was monitored in RBCs during the recovery of normal rat after phenyhydrazine treatment. Hence, the portion of membrane protein 4.1a isoform is increasing during rat RBC aging. Likewise, when the RBC life span is prolonged (but not normalised) in the b/b rats by iron-dextran treatment protein 4.1a is present in small portion in the RBC membrane. All these data indicate that the lack of protein 4.1a isoform in the b/b rat is due to the presence of young RBCs in the circulation.     

Possible involvement of Fyn kinase in ethanol-stimulated Cas tyrosine phosphorylation in rat cerebellum and cerebral cortex

In the present study, we have investigated the effect of intraperitoneal injection of ethanol (3.5 g/kg) on tyrosine phosphorylation in rat brain. Immunoblot analysis using an antiphosphotyrosine antibody revealed that a 130-kDa protein band was detected in the brain extract in response to ethanol administration. This ethanol-stimulated tyrosine phosphorylation of the 130-kDa protein was found in the brain but not in the heart, liver or thymus. The 130-kDa phosphotyrosine-containing protein was identified by immunoprecipitation to be Cas, a crk-associated src substrate. This ethanol-stimulated tyrosine phosphorylation of Cas was observed most prominently in the cerebellum and the cerebral cortex. We further examined the possible involvement of Fyn kinase in ethanol-stimulated Cas tyrosine phosphorylation. Immunecomplex kinase assay showed that Fyn was activated in the cerebellum and cerebral cortex of ethanol-administered rats. Immunoprecipitation experiments also showed that Fyn was co-immunoprecipitated with an anti-Cas antibody in these regions from ethanol-administered rats. Furthermore, exogenous Fyn was shown to phosphorylate Cas from cerebellum and cerebral cortex in vitro. These findings indicate that ethanol stimulates tyrosine phosphorylation of Cas in rat cerebellum and cerebral cortex, and that Fyn may be involved in the process.     

BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain [published erratum appears in J Cell Biol 1997 Apr 21;137(2):521]

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix of all tissues. In the mammalian central nervous system (CNS) HA is present throughout development and into adulthood. While the functions of HA are likely to be mediated by HA-binding proteins, no cell or tissue specific HA-binding proteins have been reported. In an effort to characterize the composition of the extracellular matrix of the CNS, we sought to identify neural HA-binding proteins. We report here the isolation and characterization of a cDNA with a high degree of sequence homology to members of the proteoglycan tandem repeat (PTR) family of HA-binding proteins. Unlike other HA-binding proteins, the expression of this cDNA is restricted to the CNS. We propose the name BEHAB, Brain Enriched HyAluronan Binding protein, for this gene. The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The sequence of BEHAB has long stretches of identity between rat and cat, suggesting that the encoded protein is functionally important. The size and sequence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between HA and brain proteoglycans. These observations suggest that existence of other tissue specific HA-binding proteins.     

Isoform-Specific Contributions of α-Actinin to Glioma Cell Mechanobiology

Glioblastoma Multiforme (GBM) is a malignant astrocytic tumor associated with low survival rates because of aggressive infiltration of tumor cells into the brain parenchyma. Expression of the actin binding protein α-actinin has been strongly correlated with the invasive phenotype of GBM in vivo. To probe the cellular basis of this correlation, we have suppressed expression of the nonmuscle isoforms α-actinin-1 and α-actinin-4 and examined the contribution of each isoform to the structure, mechanics, and motility of human glioma tumor cells in culture. While subcellular localization of each isoform is distinct, suppression of either isoform yields a phenotype that includes dramatically reduced motility, compensatory upregulation and redistribution of vinculin, reduced cortical elasticity, and reduced ability to adapt to changes in the elasticity of the extracellular matrix (ECM). Mechanistic studies reveal a relationship between α-actinin and non-muscle myosin II in which depletion of either α-actinin isoform reduces myosin expression and maximal cell-ECM tractional forces. Our results demonstrate that both α-actinin-1 and α-actinin-4 make critical and distinct contributions to cytoskeletal organization, rigidity-sensing, and motility of glioma cells, thereby yielding mechanistic insight into the observed correlation between α-actinin expression and GBM invasiveness in vivo.     

The Monoamine Neurons of the Rat Brain Preferentially Express a Splice Variant of α1B Subunit of the N-Type Calcium Channel

The N-type voltage-dependent calcium channels play a significant role in neurotransmitter release. The alpha1B subunit of the N-type calcium channel functions as the primary subunit that forms the pore and contains the structural motifs that mediate the pharmacological and gating properties of the channel. We report on an isoform of the alpha1B subunit that is preferentially expressed by the monoaminergic neurons of the rat brain. This isoform contains a 21-amino acid cassette in the synprint site present in the cytoplasmic loop between domains IIS6 and IIIS1. RT-PCR of micropunched tissue was used to show preferential expression of this isoform in regions of the brain containing monoaminergic neurons and to a lesser extent in the cerebellum. Double-label in situ hybridization was used to show expression of this isoform mRNA in dopaminergic neurons of the ventral mesencephalon. The expression of two distinct N-type calcium channels containing these alpha1B subunit isoforms by the monoaminergic neurons may provide for synapse-specific regulation of neurotransmitter release.     

Brevican isoforms associate with neural membranes

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.     

Expression of mitogen-activated protein kinase pathways during postnatal development of rat heart

The loss of ability to proliferate (terminal differentiation) and reduction in capability to resist ischemia are key phenomena observed during postnatal development of the heart. Mitogen-activated protein kinases (MAPKs) mediate signaling pathways for cell proliferation/differentiation and stress responses such as ischemia. In this study, the expression of these kinases and their associated kinases were investigated in rat heart ventricle. Extracts of 1-, 10-, 20-, 50-, and 365-day-old rat heart ventricles were probed with specific antibodies and their immunoreactivities were quantified by densitometry. Most of the mitogenic protein kinases including Raf1, RafB, Mek1, Erk2, and Rsk1 were significantly down-regulated, whereas the stress signaling kinases, such as Mlk3, Mekk1, Sek1, Mkk3, and Mapkapk2 were up-regulated in expression during postnatal development. Most MAP kinases including Erk1, JNKs, p38 Hog, as well as Rsk2, however, did not exhibit postnatal changes in expression. The proto-oncogene-encoded kinases Mos and Cot/Tpl 2 were up-regulated up to two- and four-fold, respectively, during development. Pak1, which may be involved in the regulation of cytoskeleton as well as in stress signaling, was downregulated with age, but the Pak2 isoform increased only after 50 days. All of these proteins, except RafB, were also detected in the isolated adult ventricular myocytes at comparable levels to those found in adult ventricle. Tissue distribution studies revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in heart, whereas the downregulated protein kinases were generally expressed in heart at relatively lesser amounts than in most of other tissues. J. Cell. Biochem. 71:286–301, 1998. © 1998 Wiley-Liss, Inc.     

Brevican-deficient mice display impaired hippocampal CA1 long-term potentiation but show no obvious deficits in learning and memory

Brevican is a brain-specific proteoglycan which is found in specialized extracellular matrix structures called perineuronal nets. Brevican increases the invasiveness of glioma cells in vivo and has been suggested to play a role in central nervous system fiber tract development. To study the role of brevican in the development and function of the brain, we generated mice lacking a functional brevican gene. These mice are viable and fertile and have a normal life span. Brain anatomy was normal, although alterations in the expression of neurocan were detected. Perineuronal nets formed but appeared to be less prominent in mutant than in wild-type mice. Brevican-deficient mice showed significant deficits in the maintenance of hippocampal long-term potentiation (LTP). However, no obvious impairment of excitatory and inhibitory synaptic transmission was found, suggesting a complex cause for the LTP defect. Detailed behavioral analysis revealed no statistically significant deficits in learning and memory. These data indicate that brevican is not crucial for brain development but has restricted structural and functional roles.     

Three Dynamin-Encoding Genes Are Differentially Expressed in Developing Rat Brain

Abstract: Dynamin proteins are members of a recently described family of GTPases involved in receptor-mediated processes. To date, three different dynamin-encoding genes have been identified in mammalian tissues. Dynamin I is expressed only in neurons, whereas dynamin II is ubiquitously expressed. A third isoform, dynamin III, was originally isolated from a rat testis cDNA library and shown to be testis-specific. However, here we report the cloning and characterization of dynamin III from brain and lung, demonstrating a more extended pattern of expression for this isoform. In addition, we have investigated the temporal pattern of expression of these three genes during brain development. We find that both dynamin I and dynamin III mRNA levels are up-regulated during embryogenesis, whereas dynamin II mRNA levels remain unchanged. From these results, we conclude that dynamin III is not a testis-specific isoform and, furthermore, that rat brain expresses three different dynamin-encoding genes that are differentially regulated during development. Therefore, this large isoform diversity of dynamin proteins in brain predicts a significant complexity in the understanding of dynamin-based processes in this tissue.