P. O. Couraud

Blood-brain barrier-specific properties of a human adult brain endothelial cell line

Establishment of a human model of the blood‐brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact‐inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up‐regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood‐brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood‐brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well‐differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Coupling of ETB endothelin receptor to mitogen-activated protein kinase stimulation and DNA synthesis in primary cultures of rat astrocytes

Abstract: Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET‐1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ETA‐R), and IRL1620, an agonist selective for the ET receptor subtype B (ETB‐R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ETB‐R is the predominant subtype in these cells. Inhibition of forskolin‐stimulated cyclic AMP production was observed under ETB‐R stimulation. Bordetella pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ETB‐R via Gi protein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen‐activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ETB‐R, but through PTX‐insensitive G protein. IRL1620‐induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf‐1. This study reveals that the various effects of ET‐1 in astrocytes are mediated by the ETB‐R, which couples to multiple signaling pathways including the MAPK cascade.

Are Several G Proteins Involved in the Different Effects of Endothelin-1 in Mouse Striatal Astrocytes?

Abstract: High‐affinity specific receptors of endothelin (ET‐1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I‐ET‐1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET‐1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide‐binding proteins.

Requirement of caveolae microdomains in extracellular signal-regulated kinase and focal adhesion kinase activation induced by Endothelin-1 in primary astrocytes

Abstract : Endothelin‐1 (ET‐1) mitogenic activity in astrocytes is mediated by the activation of the extracellular signal‐regulated kinase (ERK) pathway together with the Rho‐dependent activation of the focal adhesion kinase (FAK) pathway. To clarify the mechanisms responsible for the coordinate activation of both pathways in the ET‐1 signal propagation, the involvement of caveolae microdomains, suggested to play a role in signal transduction, was evaluated. In this study, it is reported that caveolae of primary astrocytes are enriched in endothelin receptor (ETB‐R). Furthermore, signaling molecules such as the adaptor proteins Shc and Grb2, and the small G protein Rho, also reside within these microdomains. Selective disassembly of caveolae by filipin III impairs the ET‐1‐induced tyrosine phosphorylation of proteins including ERK and FAK. In agreement with these observations, astrocytes pretreated with filipin III also failed to form stress fibers and focal adhesions and did not undergo the associated morphological changes in response to ET‐1. This study reveals that structural integrity of caveolae is necessary for the adhesion‐dependent mitogenic signals induced by ET‐1 in astrocytes, through compartmentation of ETB‐R with the upstream signaling molecules of the ERK and FAK pathways.

Functional endothelin-1 receptors in rat astrocytoma C6.

Rat astrocytoma C6 cells have been recently identified as target cells for ET-1, which stimulates inositol lipid turnover in these cells. It is shown here that binding of ET-1 to high-affinity receptors on C6 cells leads to 40-45% inhibition of isoproterenol-induced intracellular cyclic AMP accumulation, as well as to stimulation of inositol lipid turnover, both effects characterized by an absolute requirement of extracellular calcium. Moreover, ET-1, which has been generally reported to have a mitogenic effect on a variety of target cells including primary rat astrocytes, is shown here to stimulate or, alternatively, inhibit DNA synthesis in C6 cells, depending on the subclone considered.

Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-β variants in cells composing the cerebral vessel walls

Cerebral amyloid angiopathy (CAA) is an age‐associated condition and a common finding in Alzheimers disease in which amyloid‐β (Aβ) vascular deposits are featured in >80% of the cases. Familial Aβ variants bearing substitutions at positions 21–23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Aβ mutant, located outside the hot spot 21–23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endo‐thelial and smooth muscle cells with nonfibrillar structures of both variants and wild‐type Aβ40. Induction of analogous caspase‐mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan‐ and pathway‐specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Aβ peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.—Fossati, S., Cam, J., Meyerson, J., Mezhericher, E., Romero, I. A., Couraud, P. O., Weksler, B. B., Ghiso, J., Rostagno, A. Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid‐β variants in cells composing the cerebral vessel walls. FASEB J. 24, 229–241 (2010). www.fasebj.org

Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/MTUS1 in hepatocellular carcinoma.

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein (ATIP)/mitochondrial tumor suppressor gene (MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.

Antibodies raised against β-adrenergic receptors stimulate adenylate cyclase

Abstract Antibodies raised in mice against β-adrenergic receptors purified from turkey erythrocyte membranes, specifically bind to cells which possess a β-adrenergic receptor and immunoprecipitate radiolabelled purified receptor. These antibodies stimulate the adenylate cyclase activity of the turkey erythrocytes, although they do not compete with the catecholamine hormones for binding to the β-adrenergic receptor. Thus the receptor-antibody interaction, although occuring at another site than the receptor-hormone interaction, may still trigger the enzymatic activity.

Biochemical and immunochemical analysis of β-adrenergic receptor adenylate cyclase complexes

Recent years have seen an exponential growth of reports concerning the biochemical analysis of hormone and neurotransmitter receptor-effecter systems*. The studies of the various subunits of the ~etylc~li~ receptor have reached the decisive stage 01 amino acid sequence determinations as well as morphological characterization by electron microscopy. Progress has not been so rapid for the &utrenergic receptor Icyclam 51 stem. mainly because of the lack of tissue equivalent to the electric organ of tbe Torpedo tish, which contains up to tOO&Ot_t :eceptor molecules per square micron. In their search for a favorable cell, specialists 5tfthe /3-adrenergic receptors have screened animals as diverse as the frog, turkey. mouse and man, and have studied estab fished cell lines as well as fmshly recovered tissues. Despite these difiiculties. the annlysis of litc catecholamine-sensitive adenylate cyc1as.z systems has continued to attract e~rmous attention mainly because of the crucial role of catechotar ant. in human physiology attested by the avaihbility of a large variety of agonist and antagonist compounds. An equally important reason for the interest in the ~-~nergic complex is the op~~unity to study in the same system an external hormonal signal and the transmission to the inside of the cell through activation of adenylate cyclase’-‘. In this review we will summarize the latest developments in the characterization and purification of the various comprments of the ~-~~nerg~ receptor-eyclase sys tern and will present the features of the syr+ tern for which a consensus has been reached by the major investigators in the geld.

Comparison of binding characteristics of endothelin receptors on subpopulations of astrocytes

Astrocytes in primary culture originating from different brain areas of the mouse embryo (striatum, cerebral cortex and mesencephalon) were compared for their [125I]-Endothelin-1 (ET-1) binding characteristics, in terms of affinity, binding capacity and specificity. Our results indicate that astrocytes from mesencephalon express about twice as many receptors as astrocytes from striatum or cortex (149,000 +/- 9700 vs 63,700 +/- 5600 and 81,900 +/- 5300, respectively), with similar affinities. Specificity patterns for the various peptides of the endothelins/sarafotoxins family (ET-1, -2, -3; SRTXa, b, c) are comparable in the three subpopulations of astrocytes: ET-1, -2 and SRTXb exhibit higher affinities than SRTXa and SRTXc. In addition, ET-3 and SRTXc seem to discriminate between different subsets of [125I]-ET-1 binding sites in the three subpopulations.