Endothelial Cell Adhesion Molecule Expression and Lymphocyte Adhesion to Endothelial Cells: Effect of Nitric Oxide

Interactions between circulating leukocytes and vascular endothelial cells are of fundamental importance in controlling normal recirculation and migration of cells into sites of inflammation. Nitric oxide (NO), which is synthesized by vascular endothelial cells, has been reported to decrease the binding of platelets, monocytes, macrophages, and neutrophils to endothelial cells. Using NO donors and inhibitors of the enzyme NO synthase, we found no evidence that physiologically relevant levels of NO alter adhesion of purified lymphocytes to an endothelial cell line derived from human umbilical vein endothelial cells (SGHEC-7). In addition, NO donors did not alter the cell surface expression of VCAM-1, ICAM-1, or E-selectin on SGHEC-7 cells.     

Novel Cytokine-independent Induction of Endothelial Adhesion Molecules Regulated by Platelet/Endothelial Cell Adhesion Molecule (CD31)

Tumor necrosis factor–α, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell–cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule–1, and intercellular adhesion molecule–1. In contrast, ECs failed to express E-selectin when plated on poly-l-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 ± 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 ± 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co– culture of subconfluent ECs with PECAM-1– coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin–expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.The endothelial lining of the vascular system normally displays a nonactivated, nonadhesive phenotype. Stimulation with agents such as tumor necrosis factor-α (TNF-α)¹, interleukin-1 (IL-1), or lipopolysaccharide (LPS) are known to induce the expression of proteins on the endothelial surface that mediate coagulation (Bevilacqua et al., 1986), leukocyte adhesion (Bevilacqua et al., 1985; Gamble et al., 1985; Pober et al., 1986b ; Doherty et al., 1989), and leukocyte transendothelial migration (Furie et al., 1989; Moser et al., 1989). The endothelial antigens that are important for the adhesion of leukocytes are members of the selectin family, E- and P-selectin, and the immunoglobulin gene superfamily, vascular cell adhesion molecule–1 (VCAM-1) and intercellular adhesion molecule–1 (ICAM-1) (Carlos and Harlan, 1994; Litwin et al., 1995).The induction of E-selectin expression on endothelial cells (ECs) in vitro after cytokine stimulation is transient and independent of the continued presence of the stimulant (Pober et al., 1986a ). Previous studies have shown that E-selectin mRNA and protein levels peak between 2 and 4 h, respectively, after treatment with an agonist, returning to near basal levels by 24 h (Bevilacqua et al., 1989; Read et al., 1994). VCAM-1 (Osborn et al., 1989) and ICAM-1 (Pober et al., 1986b ) are maximal 6 and 12 h, respectively, after stimulation.In contrast to the transiency of E-selectin and VCAM expression demonstrated by the in vitro data, these antigens have been detected on venular endothelium in chronic inflammatory lesions, such as the synovium in rheumatoid arthritis (Koch et al., 1991), and the skin in psoriasis (Petzelbauer et al., 1994). E-selectin expression is also detected on angiogenic vessels in human hemangiomas, a noninflammatory angiogenic disease (Kraling et al., 1996). Moreover, the architecture and anatomic localization of capillary loops influence the pattern of endothelial expression of E-selectin and VCAM-1, independently of the availability of cytokines (Petzelbauer et al., 1994). Thus it is likely that alternate control mechanisms exist to allow prolonged, locality-based expression of adhesion molecules on the endothelium. At least one of these alternate mechanisms may be flow, since increased shear stress has been shown to selectively modulate adhesion molecule expression, upregulating ICAM-1 but not E-selectin or VCAM-1 (Nagel et al., 1994).Since sites of inflammation are often associated with morphological changes including cell retraction of the endothelium (Schumacher, 1973), we hypothesized that cell contacts may be important in the regulation of endothelial phenotype. We describe here the central role of the junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1), through the formation of cell–cell interactions, in the maintenance of the functional integrity of the endothelial monolayer. Furthermore, we demonstrate a novel pathway for the induction of adhesion molecules on endothelial cells that is independent of exogenous addition of cytokines, but is related to integrin- and cell shape–associated signaling events.     

Bacterial Adhesion under Static and Dynamic Conditions

The deposition of various pseudomonads and coryneform bacteria with different hydrophobicities (water contact angles) and negative cell surface charges on negatively charged Teflon and glass surfaces was investigated. The levels of deposition varied between 5.0 × 10⁴ and 1.6 × 10⁷ cells cm^-2 and between 5.0 × 10⁴ and 3.6 × 10⁷ cells cm^-2 for dynamic column and static batch systems, respectively, indicating that there was a wide variation in physicochemical interactions. Batch and column results were compared in order to better distinguish between hydrodynamic and other system-dependent influences and method-independent physicochemical interactions. Despite the shorter suspension-solid contact time in columns (1 h) than in batch systems (4 h), the level of deposition (expressed as the number of cells that adhered) divided by the applied ambient cell concentration was 4.12 ± 1.63 times higher in columns than in batch sytems for 15 of 22 strain-surface combinations studied. This demonstrates that transport of microbial particles from bulk liquid to surfaces is more efficient in dynamic columns (transport dominated by convection and diffusion) than in static batch systems (transport by diffusion only). The relative constancy of this ratio for the 15 combinations shows that physicochemical interactions affect adhesion similarly in the two systems. The deviating deposition behavior of the other seven strain-surface combinations could be attributed to method-dependent effects resulting from specific cell characteristics (e.g., to the presence of capsular polymers, to an ability to aggregate, to large cell sizes, or to a tendency to desorb after passage through an air-liquid interface).     

Adhesion Between Human Neutrophils and Immobilized Endothelial Ligand Vascular Cell Adhesion Molecule 1: Divalent Ion Effects

Integrin-mediated adhesion of circulating neutrophils to endothelium during inflammation involves multiple adhesion molecules on both neutrophils and endothelium. Most studies of neutrophil adhesion have focused on adhesion to ICAM-1 (mediated by β₂ integrins), but interaction with the endothelial ligand vascular cell adhesion molecule 1 (VCAM-1) may also play a role in neutrophil adhesion to activated endothelium. In this study we demonstrate significant adhesion between neutrophils and VCAM-1 mediated by β₁ integrins, principally via α₄β₁ (VLA-4). We characterize the dynamics of adhesion in terms of rate constants for a two-step bond formation process, the first involving juxtaposition of active molecules with substrate and the second involving bond formation. The results indicate that the first step is rate limiting for VLA-4-VCAM-1 interactions. Changing divalent cation composition affects these coefficients, implicating molecular conformational changes as a key step in the process.     

Changes in the Level of Neuronal Cell Adhesion Molecule in the Brain of Male Rats under Conditions of Suppression of Production of Testosterone

Under conditions of experimental varicocele in rats, we observed suppression of production of testosterone and significant drops in the level of this hormone in the blood serum. Such a decrease resulted in a two-fold (or even greater) rise in the amount of the membrane-bound fraction of neuronal cell adhesion molecule (NCAM) in the hypothalamus and hippocampus, while the levels in the cerebellum and neocortex remained stable, and also in appreciable redistribution of the soluble form of this molecule in different cerebral structures in the course of development of varicocele.     

SOD3 Reduces Inflammatory Cell Migration by Regulating Adhesion Molecule and Cytokine Expression

Inflammatory cell migration characteristic of ischemic damages has a dual role providing the tissue with factors needed for tissue injury recovery simultaneously causing deleterious development depending on the quality and the quantity of infiltrated cells. Extracellular superoxide dismutase (SOD3) has been shown to have an anti-inflammatory role in ischemic injuries where it increases the recovery process by activating mitogen signal transduction and increasing cell proliferation. However, SOD3 derived effects on inflammatory cytokine and adhesion molecule expression, which would explain reduced inflammation in vascular lesions, has not been properly characterized. In the present work the effect of SOD3 on the inflammatory cell extravasation was studied in vivo in rat hind limb ischemia and mouse peritonitis models by identifying the migrated cells and analyzing SOD3-derived response on inflammatory cytokine and adhesion molecule expression. SOD3 overexpression significantly reduced TNFα, IL1α, IL6, MIP2, and MCP-1 cytokine and VCAM, ICAM, P-selectin, and E-selectin adhesion molecule expressions in injured tissues. Consequently the mononuclear cell, especially CD68+ monocyte and CD3+ T cell infiltration were significantly decreased whereas granulocyte migration was less affected. According to our data SOD3 has a selective anti-inflammatory role in ischemic damages preventing the migration of reactive oxygen producing monocyte/macrophages, which in excessive amounts could potentially further intensify the tissue injuries therefore suggesting potential for SOD3 in treatment of inflammatory disorders.     

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.     

Inside-out Signaling Promotes Dynamic Changes in the Carcinoembryonic Antigen-related Cellular Adhesion Molecule 1 (CEACAM1) Oligomeric State to Control Its Cell Adhesion Properties

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) can engage in both cis-homophilic (parallel) oligomerization and trans-homophilic (anti-parallel) binding. In this study, we establish that the CEACAM1 transmembrane domain has a propensity to form cis-dimers via the transmembrane-embedded ⁴³²GXXXG⁴³⁶ motif and that this basal state is overcome when activated calmodulin binds to the CEACAM1 cytoplasmic domain. Although mutation of the ⁴³²GXXXG⁴³⁶ motif reduced CEACAM1 oligomerization, it did not affect surface localization of the receptor or influence CEACAM1-dependent cellular invasion by the pathogenic Neisseria. The mutation did, however, have a striking effect on CEACAM1-dependent cellular aggregation, increasing both the kinetics of cell-cell association and the size of cellular aggregates formed. CEACAM1 association with tyrosine kinase c-Src and tyrosine phosphatases SHP-1 and SHP-2 was not affected by the ⁴³²GXXXG⁴³⁶ mutation, consistent with their association with the monomeric form of wild type CEACAM1. Collectively, our results establish that a dynamic oligomer-to-monomer shift in surface-expressed CEACAM1 facilitates trans-homophilic binding and downstream effector signaling.     

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.     

Soluble Melanoma Cell Adhesion Molecule (sMCAM/sCD146) Promotes Angiogenic Effects on Endothelial Progenitor Cells through Angiomotin

The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.     

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α₄β₁ integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels ∼2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10⁻⁷ M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.     

盘基网柄菌细胞黏附分子cadA/DdCAD-1在细胞分化及细胞命运决定中的作用

盘基网柄菌细胞黏附分子DdCAD-1是在细胞发育过程中最先表达的黏附分子,为了研究DdCAD-1在盘基网柄菌细胞发育中的作用,将cadA基因的突变株cadA-细胞用中性红染料染色,发育成的蛞蝓体显示cadA-细胞的前柄细胞/前孢子细胞的分化出现明显的障碍,外源表达的重组蛋白His6-DdCAD-1与cadA-细胞作用一段时间后,这种现象得到了改善。另外,cadA-细胞的孢子产率也有所降低,外源重组蛋白也可以拯救该表现型。表达DdCAD-1的细胞与cadA-细胞共同发育所形成的嵌合体显示表达DdCAD-1的细胞占据在拔顶期结构的顶端及尾部,而这些结构都在非孢子区,最终会死亡。提示DdCAD-1对于细胞分化及细胞命运决定有重要意义。     

Expression of Endothelial Cell Adhesion Molecules in Joints and Heart during Borrelia burgdorferi Infection of Mice

The expression of adhesion molecules on endothelia was examined during chronic arthritis and carditis in SCID and immunocompetent susceptible AKR/N mice infected with Borrelia burgdorferi (B. burgdorferi). All stages of disease were associated with the upregulation or new expression of ICAM-1 and P-selectin and of VCAM-1 and E-selectin, respectively, on blood vessels of affected joint tissues of SCID and AKR/N mice as well as on heart tissue of SCID mice but not in other tissues. Moreover, ICAM-1 was also found on infiltrating mononuclear cells. The overall staining intensity for each of the four adhesion molecules on individual tissue sections of joint and heart increased with time of infection and was associated with the presence of spirochetes in the tissue. In addition it is shown that in both mouse strains inflammation of joints but not heart is accompanied by vascular proliferation. Synovial but not heart tissues of infected SCID mice were found to express both, peripheral-(PNAd) and mucosal (MAdCAM-1) lymph node high endothelia venule associated vascular addressins as detected by mAb Meca-79 and Meca-367, respectively, but only at later stages of the disease and only on newly generated small venules. However, neither of the two addressins were evident in synovial lesions of AKR/N mice. Together the data suggest that the concomittant induction of ICAM-1, VCAM-1, E-selectin and P-selectin in lesions of infected mice provide a means for enhanced cellular infiltration into affected organs and that the regulation of these structures is conserved in the absence of a functional immune system. Furthermore, the differential induction of vascular proliferation in joint and heart tissues as well as the restricted expression patterns of vascular addressins indicate that the pathogenetic processes induced by B. burgdorferi are distinct for joint and heart.     

Transforming Growth Factor β1-Regulated Cell Proliferation and Expression of Neural Cell Adhesion Molecule in B104 Neuroblastoma Cells

The expression and activity of factors influencing early neuronal development are altered by ethanol. Such factors include growth factors, for example, platelet-derived growth factor and basic fibroblast growth factor (for cell proliferation), and cell adhesion molecules (for neuronal migration). One agent, transforming growth factor beta1 (TGFbeta1), may affect both events. We tested the hypothesis that ethanol alters myriad TGFbeta1-mediated activities [i.e., cell proliferation and neural cell adhesion molecule (N-CAM) expression] using B104 neuroblastoma cells. TGFbeta1 inhibited the proliferation of B104 cells as evidenced by decreases in cell number and [3H]thymidine ([3H]dT) incorporation. TGFbeta1 induced sustained activation of extracellular signal-regulated kinases (ERKs), which are part of the family of mitogen-activated protein kinases (MAPKs). Treatment with PD98059 (a MAPK kinase blocker) abolished TGFbeta1-regulated inhibition of [3H]dT incorporation. TGFbeta1-mediated growth inhibition was potentiated by ethanol exposure. Ethanol also produced prolonged activation of ERK, an effect that was partially eliminated by treatment with PD98059. On the other hand, TGFbeta1 up-regulated N-CAM expression, and this up-regulation was not affected by treatment with PD98059. Ethanol inhibited the TGFbeta1-induced up-regulation of N-CAM expression in a concentration-dependent manner. Thus, TGFbeta1 affects ERK-dependent cell proliferation and ERK-independent N-CAM expression in B104 cells. Both activities are sensitive to ethanol and may underlie the ethanol-induced alterations in the proliferation and migration of CNS neurons.