Transforming Growth Factor-beta Response Research Articles

Preeclampsia: novel insights from global RNA profiling of trophoblast subpopulations

The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis. Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls. This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non-protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization. Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type-specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia andthose that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the syncytiotrophoblast data highlighted the dysregulation of immune functions, morphogenesis, transport, and responses to vascular endothelial growth factor and progesterone. The invasive cytotrophoblast data provided evidence of alterations in cellular movement, which is consistent with the shallow invasion often associated with severe preeclampsia. Other dysregulated pathways included immune, lipid, oxygen, and transforming growth factor-beta responses. The data for endovascular cytotrophoblasts showed disordered metabolism, signaling, and vascular development. Additionally, the transcriptional data revealed the differential expression in severe preeclampsia of 2 classes of non-coding RNAs: long non-coding RNAs and small nucleolar RNAs. The long non-coding RNA, urothelial cancer associated 1, was the most highly up-regulated in this class. In situ hybridization confirmed severe preeclampsia-associated expression in syncytiotrophoblasts. The small nucleolar RNAs, which chemically modify RNA structure, also correlated with severe preeclampsia. Thus, we enumerated Cajal body foci, sites of small nucleolar RNA activity, in primary cytotrophoblasts that were isolated from control and severe preeclampsia placentas. In severe preeclampsia, cytotrophoblasts had approximately double the number of these foci as the control samples. A laser microdissection approach enabled the identification of novel messenger RNAs and non-coding RNAs that were misexpressed by various trophoblast subpopulations in severe preeclampsia. The results suggested new avenues of investigation, in particular, the roles of PRG2, Kell blood group determinants, and urothelial cancer associated 1 in syncytiotrophoblast diseases. Additionally, many of the newly identified dysregulated molecules might have clinical utility as biomarkers of severe preeclampsia.

Different European-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs

Immunization of piglets with two different European-type modified live vaccines against porcine reproductive and respiratory syndrome (PRRS) virus produced different outcomes. After vaccination, pigs became viremic (42 days), neutralizing antibodies did not develop, and frequencies of virus-specific gamma-interferon-secreting cells (IFN-γ-SC) were low. Levels of interleukin-10 (IL-10) produced by peripheral blood mononuclear cells (PBMC) seemed to inversely correlate with interferon-gamma responses. After a challenge with a virulent Spanish strain, one vaccine (V3) protected piglets against viremia while the other (V1) did not. The vaccine V3 induced the highest IFN-γ-SC frequencies. IL-2, IL-4 or transforming growth factor-beta responses were not detected at any time for neither of the vaccines. In contrast, haptoglobin rose in sera of viremic pigs after the challenge. These results indicated a strong involvement of IFN-γ, and maybe IL-10, in the development of immunity against PRRS virus.

RUNX3 inhibits cell proliferation and induces apoptosis by reinstating transforming growth factor beta responsiveness in esophageal adenocarcinoma cells

BackgroundSEG-1, a Barrett's-derived esophageal adenocarcinoma cell line, is not responsive to transforming growth factor beta (TGF-β) growth effects. We hypothesize that SEG-1 cells lack the tumor-suppressor gene Runt domain transcription factor 3 (RUNX3) and that its reinstatement can restore the antiproliferative and apoptotic effects of TGF-β. MethodsRUNX3 expression was assessed by immunoblotting. SEG-1 cells were transfected with RUNX3 and treated with TGF-β. The effects of RUNX3 transfection on cell proliferation and apoptosis were determined. Smad-mediated TGF-β transcriptional activity was evaluated with the use of dual-luciferase assay. ResultsSEG-1 cells are not responsive to TGF-β. SEG-1 cells lack RUNX3 protein expression, while RUNX3 is highly expressed in normal human gastric and esophageal epithelium. Although the Smad-2 signaling is activated by TGF-β, SEG-1 cells lack Smad-mediated TGF-β transcriptional activity. In cells transfected with RUNX3, TGF-β acquired an antiproliferative effect and induced apoptosis (P=.001). RUNX3 transfection, in the absence of TGF-β, had no effect on proliferation and apoptosis of SEG-1 cells. RUNX3 expression dramatically increases SMAD-mediated TGF-β–induced transcriptional activity when compared with controls (P=.0001). ConclusionsRUNX3 is not expressed in SEG-1 cells, while it is present in normal esophageal mucosa. RUNX3 is essential for the antiproliferative and apoptotic effects of TGF-β in SEG-1 cells and for the Smad-mediated transcriptional activity of TGF-β.

Transforming growth factor beta regulates cell-cell adhesion through extracellular matrix remodeling and activation of focal adhesion kinase in human colon carcinoma Moser cells.

Transforming growth factor (TGF) beta is a potent regulator of cell-matrix and cell-cell adhesions (collectively termed cellular adhesions). Cellular adhesions play crucial roles in controlling the differentiation of epithelial cells and in maintaining the integrity of the epithelium. Loss of TGF beta-responsiveness is thought to be an important early initiating event in the malignant progression of epithelial cancer. In the TGFbeta-responsive human colon adenocarcinoma Moser cells, TGFbeta promotes cellular adhesions and suppresses their malignant phenotype. TGFbeta promotes cell-matrix adhesion by inducing the synthesis of extracellular matrix (ECM) adhesion molecules and the expression of integrin receptors for these molecules (termed ECM remodeling). TGFbeta promotes cell-cell adhesion through the induction of E-cadherin expression, an epithelial associated homotypic cell-cell adhesion molecule, which also functions as a tumor suppressor in colon cancer. How TGFbeta regulates E-cadherin expression is not known. In this study, we showed that the induction of E-cadherin by TGFbeta was mediated through the activation of focal adhesion kinase (FAK), a major signaling molecule in focal adhesion contacts and that the activation of FAK was due to ECM remodeling and increased cell-matrix interactions. Thus, TGFbeta regulates cell-cell adhesion through its ability to remodel the ECM and to activate FAK through ECM remodeling.

Sustained Activation of Fibroblast Transforming Growth Factor-β/Smad Signaling in a Murine Model of Scleroderma

Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the possibility that Smads plays an important part in the pathogenesis of fibrosis, and may therefore represent targets for selective anti-fibrotic interventions.

Role of p38 MAPK in Transforming Growth Factor β Stimulation of Collagen Production by Scleroderma and Healthy Dermal Fibroblasts

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.

The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization.

Smad proteins mediate the transforming growth factor beta responses. C-terminal phosphorylation of R-Smads leads to the recruitment of Smad4 and the formation of active signaling complexes. We investigated the mechanism of phosphorylation-induced Smad complex formation with an activating pseudo-phosphorylated Smad3. Pseudo-phosphorylated Smad3 has a greater propensity to homotrimerize, and recruits Smad4 to form a heterotrimer containing two Smad3 and one Smad4. The trimeric interaction is mediated through conserved interfaces to which tumorigenic mutations map. Furthermore, a conserved Arg residue within the L3 loop, located near the C-terminal phosphorylation sites of the neighboring subunit, is essential for trimerization. We propose that the phosphorylated C-terminal residues interact with the L3 loop of the neighboring subunit to stabilize the trimer interaction.

A sense phosphorothioate oligodeoxynucleotide containing the transforming growth factor beta regulatory element acts as a novel local nonsteroidal antifibrotic drug

Fibrosis is a potential response to tissue injury. At present, glucocorticoids with their numerous toxic side effects are the only effective treatment for fibrotic diseases. Granulomas induced by sponge implantation were treated with single-stranded phosphorothioate oligodeoxynucleotides containing the wild type or mutated transforming growth factor-beta response element designed to inhibit the rat proalpha1(I) promoter activity. Single-stranded phosphorothioate oligonucleotides resulted in antifibrotic activity based on their ability to reduce granuloma tissue formation and selectively inhibit collagen synthesis. The mutated single-standed phosphorothioate oligonucleotides or dexamethasone given at an equivalent dose to single-standed phosphorothioate oligonucleotides failed to do so. These data suggest that the phosphorothioate oligodeoxynucleotide containing the transforming growth factor-beta regulatory element has an antifibrotic effect and may be used to inhibit the development of fibrosis.

Major histocompatibility complex-controlled protective influences on experimental autoimmune encephalomyelitis are peptide specific.

The myelin basic protein (MBP) peptide 63-88-induced experimental autoimmune encephalomyelitis (EAE) and its associated T cell cytokine profile are influenced by the rat major histocompatibility complex (MHC). There is an allele-specific protective influence of the MHC class I region, whereas the MHC class II region display either disease-protective or -promoting effects. To investigate if the MHC-associated protection is dependent on certain combinations of MBP peptide and MHC molecules, we have now used another peptide (MBP 89-101). A broader and different set of rat MHC alleles were associated with EAE induced with MBP 89-101 as compared to MBP 63-88. All EAE-susceptible strains mounted peptide-specific strong T helper (Th) 1-like immune responses in vitro. Immunization of rats with an extended peptide (MBP 87-110) induced EAE associated with the same MHC haplotypes as the 89-101 peptide, except in LEW.1N (RT1 pi) rats which were relatively resistant. Only this strain responded with additional Th2-like and transforming growth factor-beta responses to the peptide in vitro. In vivo depletion of CD8+ cells aggravated the disease in this strain. We conclude that both MHC-controlled promoting and protective influences on EAE are dependent on certain MHC/MBP peptide combinations, and that the 87-110 region of MBP contains a major MHC-associated encephalitogenic epitope in the rat.

Differentiation of human trophoblast cells in vitro is inhibited by dimethyl sulfoxide

The maternal signs of preeclampsia, which include the new onset of high blood pressure, can occur because of faulty placentation. We theorized that transcriptomic analyses of trophoblast subpopulations in situ would lend new insights into the role of these cells in preeclampsia pathogenesis.Our goal was to enrich syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts from the placentas of severe preeclampsia cases. Total RNA was subjected to global transcriptional profiling to identify RNAs that were misexpressed compared with controls.This was a cross-sectional analysis of placentas from women who had been diagnosed with severe preeclampsia. Gestational age-matched controls were placentas from women who had a preterm birth with no signs of infection. Laser microdissection enabled enrichment of syncytiotrophoblasts, invasive cytotrophoblasts, or endovascular cytotrophoblasts. After RNA isolation, a microarray approach was used for global transcriptional profiling. Immunolocalization identified changes in messenger RNA expression that carried over to the protein level. Differential expression of non–protein-coding RNAs was confirmed by in situ hybridization. A 2-way analysis of variance of non-coding RNA expression identified particular classes that distinguished trophoblasts in cases vs controls. Cajal body foci were visualized by coilin immunolocalization.Comparison of the trophoblast subtype data within each group (severe preeclampsia or noninfected preterm birth) identified many highly differentially expressed genes. They included molecules that are known to be expressed by each subpopulation, which is evidence that the method worked. Genes that were expressed differentially between the 2 groups, in a cell-type–specific manner, encoded a combination of molecules that previous studies associated with severe preeclampsia and those that were not known to be dysregulated in this pregnancy complication. Gene ontology analysis of the syncytiotrophoblast data highlighted the dysregulation of immune functions, morphogenesis, transport, and responses to vascular endothelial growth factor and progesterone. The invasive cytotrophoblast data provided evidence of alterations in cellular movement, which is consistent with the shallow invasion often associated with severe preeclampsia. Other dysregulated pathways included immune, lipid, oxygen, and transforming growth factor-beta responses. The data for endovascular cytotrophoblasts showed disordered metabolism, signaling, and vascular development. Additionally, the transcriptional data revealed the differential expression in severe preeclampsia of 2 classes of non-coding RNAs: long non-coding RNAs and small nucleolar RNAs. The long non-coding RNA, urothelial cancer associated 1, was the most highly up-regulated in this class. In situ hybridization confirmed severe preeclampsia-associated expression in syncytiotrophoblasts. The small nucleolar RNAs, which chemically modify RNA structure, also correlated with severe preeclampsia. Thus, we enumerated Cajal body foci, sites of small nucleolar RNA activity, in primary cytotrophoblasts that were isolated from control and severe preeclampsia placentas. In severe preeclampsia, cytotrophoblasts had approximately double the number of these foci as the control samples.A laser microdissection approach enabled the identification of novel messenger RNAs and non-coding RNAs that were misexpressed by various trophoblast subpopulations in severe preeclampsia. The results suggested new avenues of investigation, in particular, the roles of PRG2, Kell blood group determinants, and urothelial cancer associated 1 in syncytiotrophoblast diseases. Additionally, many of the newly identified dysregulated molecules might have clinical utility as biomarkers of severe preeclampsia.

The type II transforming growth factor-beta receptor as a tumor-suppressor gene.

Recent work has shown that: 1) loss of transforming growth factor-beta response is associated with malignant progression, 2) maintenance of autocrine negative transforming growth factor-beta activity is a key impediment to malignant progression, and 3) the major mechanism for loss of RII expression in replication error-positive colorectal cancer patients is mutation of the poly A tract of the transforming growth factor-beta receptor type II (RII) gene resulting in the generation of a premature STOP codon. Major issues for the role of RII in cancer are identified as the determination of the penetrance of mechanisms of RII loss in non-replication error tumors and other types of malignancies in addition to colon cancer. Analysis of mechanism of RII loss may prove to have clinical use in defining the clinical course of subset of different types of malignancies and, in addition, it may result in the identification of new therapeutic targets and approaches for some subsets of cancers.

Glucocorticoids coordinately regulate type I collagen pro alpha 1 promoter activity through both the glucocorticoid and transforming growth factor beta response elements: a novel mechanism of glucocorticoid regulation of eukaryotic genes.

Glucocorticoids have previously have shown to decrease Type I collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type I procollagen gene expression. These latter studies have included uridine incorporation into pro alpha 1 (I) and pro alpha 2 (I) mRNAs and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking region of the pro alpha 1 (I) collagen gene and the reporter gene, chloramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibroblasts that dexamethasone down regulates the promoter activity of the pro alpha 1 (I) collagen gene. The glucocorticoid-mediated down-regulation of procollagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whole ColCat 3.6 plasmid did not eliminate the effect. The possibility existed that another cis-element in the 5' flanking region of the pro alpha 1 (I) collagen gene was also required for the collagen glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate in a decrease of transforming growth factor-beta (TGF-beta) secretion into the media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid receptor binding to the GRE and TGF-beta activator protein to the TGF-beta element which were brought back to control values by coordinate exogenous TGF-beta treatment. Thus the interaction of these TGF-beta molecules with cellular membrane receptors and subsequent transduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expression through both the GRE and TGF-beta elements. Depression of procollagen gene expression by glucocorticoids through the TGF-beta element is mediated by decreased TGF-beta secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the basis for a novel mechanism of glucocorticoid-mediator regulation of eukaryotic genes containing the TGF-beta element.

Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1

Skeletal alpha-actin (SkA) is representative of the cardiac genes that are expressed at high levels in embryonic myocardium, down-regulated after birth, and reactivated by trophic signals including type beta-transforming growth factors (TGF beta). To investigate the molecular basis for cardiac-restricted and TGF beta-induced SkA transcription, we have undertaken a mutational analysis of the SkA promoter in ventricular myocytes, with emphasis on the role of three nominal serum response elements. Serum response factor (SRF) and the bifunctional factor YY1 are the predominant cardiac proteins contacting the proximal SRE (SRE1). Mutations of SRE1 that prevent recognition by SRF and YY1, or SRF alone, virtually abolish SkA transcription in both TGF beta- and vehicle-treated cells; mutation of distal SREs was ineffective. A mutation which selectively abrogates YY1 binding increases both basal and TGF beta-dependent expression, substantiating the predicted role of YY1 as an inhibitor of SRF effects. However, efficient SkA transcription requires combinatorial action of SRE1 with consensus sites for Sp1 and the SV40 enhancer-binding protein, TEF-1. As isolated motifs, either SRE1- or TEF-1-binding sites function as TGF beta response elements. Induction of the SkA promoter by TGF beta required SRF and TEF-1 in concert, unlike other pathways for TGF beta-dependent gene expression.

Bleomycin stimulates pro-alpha 1 (I) collagen promoter through transforming growth factor beta response element by intracellular and extracellular signaling.

The role of transforming growth factor beta as a mediator of the fibrogenic effect of bleomycin in lung has been investigated at the transcriptional level. Several constructs containing the rat pro-alpha 1 (I) collagen promoter fused to the chloramphenicol acetyltransferase gene were transfected into rat lung fibroblasts. Both bleomycin and transforming growth factor beta 1 increased promoter activity in fibroblasts transfected with constructs containing the transforming growth factor beta response element. Fibroblasts transfected with a deletion construct that lacks this response element did not respond to either bleomycin or transforming growth factor beta 1. Anti-transforming growth factor beta 1-neutralizing antibodies did not block the increase in promoter activity induced by bleomycin, suggesting intracellular signaling. Mutation of the transforming growth factor beta response element greatly reduced the bleomycin effect, which also infers intracellular signaling. In addition, plasmin added to the media greatly enhanced bleomycin stimulation of promoter activity demonstrating that transforming growth factor beta mediates the bleomycin effect through extracellular signaling.

Isolation and characterization of transforming growth factor beta response variants from human prostatic tumor cell lines.

In this study we examined the relation between the response to transforming growth factor beta (TGF beta 1) in vitro and the growth in vivo of 1-LN-PC3-1A (1-LN) human prostatic carcinoma cells. 1-LN cells resistant to the growth-inhibitory effects of TGF beta 1 were isolated after exposure to 2 ng/ml TGF beta 1 in an anchorage-independent growth assay. Cloning of TGF beta 1-resistant and -sensitive populations produced 2 clones (R2-6 and 1-LN clone 4), which maintained relatively stable resistance or sensitivity, respectively, in the absence of TGF beta 1 for up to 12 passages. Colony formation by the R2-6 cells in the presence of TGF beta 1 was 2-10 times greater than that of 1-LN clone 4, depending upon the TGF beta 1 concentration. Injection of 1 x 10(5) R2-6 cells into athymic nude mice produced tumors with a significantly shorter latency interval as compared with 1-LN clone 4 tumors (P < 0.0001). Western immunoblotting showed that higher levels of latent TGF beta 1 protein were secreted into the culture medium by 1-LN clone 4 cells. Acidified conditioned media from both clones inhibited mink lung epithelial cell DNA synthesis. Neutralizing monoclonal antibody to TGF beta 1 but not TGF beta 2 abrogated this inhibitory effect. Comparison of the different sensitive and resistant clones showed that in vitro sensitivity to TGF beta 1 and in vivo tumor latency interval were not invariably correlated. Thus, the TGF beta 1 response phenotype in vitro was not always predictive of growth delay in vivo.

Epstein-Barr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity.

A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.