Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells.

Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density-dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density-dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low-density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp -650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells.

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

The nucleation of receptor-mediated endocytosis.

A theory of the mechanical origins of receptor-mediated endocytosis shows that a spontaneous membrane complex formation can provide the stimulus for a local membrane motion toward the cytosol. This motion is identified with a nucleation stage of receptor-mediated endocytosis. When membrane complexes cluster, membrane deformation is predicted to be most rapid. The rate of growth of membrane depressions depends upon the relative rates of approach of aqueous cytosolic and extracellular fluids toward the cell membrane. With cytosolic and extracellular media characterized by apparent viscosities, the rate of growth of membrane depressions is predicted to increase as the extracellular viscosity nears the apparent viscosity of the cytosol and then to decrease when the extracellular viscosity exceeds that of the cytosol. To determine whether these trends would be apparent in the overall endocytosis rate constant, an experimental study of transferrin-mediated endocytosis in two different cell lines was conducted. The experimental results reveal the same dependence of internalization rate on extracellular viscosity as predicted by the theory. These and other comparisons with experimental data suggest that the nucleation stage of receptor-mediated endocytosis is important in the overall endocytosis process.

Arrest of endotoxin-induced hypotension by transforming growth factor beta1.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.

The conditioning of culture medium by the production of growth-regulatory substances is a well-established phenomenon with eukaryotic cells. It has recently been shown that many prokaryotes are also capable of modulating growth, and in some cases sensing cell density, by production of extracellular signaling molecules, thereby allowing single celled prokaryotes to function in some respects as multicellular organisms. As Escherichia coli shifts from exponential growth to stationary growth, many changes occur, including cell division leading to formation of short minicells and expression of numerous genes not expressed in exponential phase. An understanding of the coordination between the morphological changes associated with cell division and the physiological and metabolic changes is of fundamental importance to understanding regulation of the prokaryotic cell cycle. The ftsQA genes, which encode functions required for cell division in E. coli, are regulated by promoters P1 and P2, located upstream of the ftsQ gene. The P1 promoter is rpoS-stimulated and the second, P2, is regulated by a member of the LuxR subfamily of transcriptional activators, SdiA, exhibiting features characteristic of an autoinduction (quorum sensing) mechanism. The activity of SdiA is potentiated by N-acyl-homoserine lactones, which are the autoinducers of luciferase synthesis in luminous marine bacteria as well as of pathogenesis functions in several pathogenic bacteria. A compound(s) produced by E. coli itself during growth in Luria Broth stimulates transcription from P2 in an SdiA-dependent process. Another substance(s) enhances transcription of rpoS and (perhaps indirectly) of ftsQA via promoter P1. It appears that this bimodal control mechanism may comprise a fail-safe system, such that transcription of the ftsQA genes may be properly regulated under a variety of different environmental and physiological conditions.

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.

A mutant p53 antagonizes the deregulated c-myc-mediated enhancement of apoptosis and decrease in leukemogenicity.

Myeloid leukemic M1 cells that do not express p53 and transfected M1 clones that constitutively express the [Val135]p53 mutant or deregulated c-myc or coexpressing both genes grew autonomously in culture with a similar growth rate and cloning efficiency. Expression of deregulated c-myc in M1 leukemic cells enhanced susceptibility to induction of apoptotic cell death and resulted in a reduced leukemogenicity when injected into isologous mice. Expression of the [Val135]p53 mutant did not change cell susceptibility to induction of apoptosis or leukemogenicity, but expression of this mutant p53 suppressed the effects of deregulated c-myc on these properties. The results indicate that the [Val135]p53 mutant can show a gain of function for susceptibility to apoptosis and leukemogenicity in leukemic cells with deregulated c-myc and, thus, enhance tumor development.

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

Drosophila hormone receptor 38: a second partner for Drosophila USP suggests an unexpected role for nuclear receptors of the nerve growth factor-induced protein B type.

In Drosophila the response to the hormone ecdysone is mediated in part by Ultraspiracle (USP) and ecdysone receptor (EcR), which are members of the nuclear receptor superfamily. Heterodimers of these proteins bind to ecdysone response elements (EcREs) and ecdysone to modulate transcription. Herein we describe Drosophila hormone receptor 38 (DHR38) and Bombyx hormone receptor 38 (BHR38), two insect homologues of rat nerve growth factor-induced protein B (NGFI-B). Although members of the NGFI-B family are thought to function exclusively as monomers, we show that DHR38 and BHR38 in fact interact strongly with USP and that this interaction is evolutionarily conserved. DHR38 can compete in vitro against EcR for dimerization with USP and consequently disrupt EcR-USP binding to an EcRE. Moreover, transfection experiments in Schneider cells show that DHR38 can affect ecdysone-dependent transcription. This suggests that DHR38 plays a role in the ecdysone response and that more generally NGFI-B type receptors may be able to function as heterodimers with retinoid X receptor type receptors in regulating transcription.

Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin.

Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization.

Tumorigenic activity of the BCR-ABL oncogenes is mediated by BCL2.

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p210BCR-ABL and p190BCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCR-ABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation.