Involvement of phosphoinositide 3-kinase and Rac in membrane ruffling induced by IL-2 in T cells

IL-2 is known to play a critical role in regulating T lymphocyte proliferation. We show here that IL-2 also provokes an instantaneous and sustained membrane ruffling in cloned human or murine T cells as well as in lectin-activated peripheral blood lymphocytes. In the IL-2-induced lamellipodia, tubulin is depolymerized whereas actin is strongly polymerized, forming caps. IL-2-induced membrane ruffling is protein kinase C (PKC) independent, as judged by the absence of effects of bisindolylmaleimide, an efficient inhibitor of all PKC isoforms. The formation of lamellipodia by IL-2 is blocked by wortmannin and LY294002, two inhibitors of phosphoinositide 3-kinase (PI3-kinase). Moreover, expression in murine T cells of an inactive form of P13-kinase inhibits IL-2-induced membrane ruffling, whereas expression of a constitutively active p110 increases the basal membrane ruffling. Rac is also involved in IL-2-induced membrane ruffling since an inactive form of Rac (N17rac) blocks the IL-2-induced lamellipodia, whereas the constitutive form of Rac (Val12rac) can also lead to membrane ruffling. In the signaling cascade, Rac is downstream of PI3-kinase since constitutive membrane ruffling in Val12rac cells is insensitive to wortmannin. Thus, through a signaling cascade involving PI3-kinase and Rac, IL-2 can induce profound alterations of the T cell cytoskeleton, a phenomenon which might be of importance for T cell physiology.     

Inhibition of invasion of epithelial cells by Tiam1-Rac signaling

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.     

A role for perlecan in the suppression of growth and invasion in fibrosarcoma cells

Perlecan is a major heparan sulfate proteoglycan of basement membranes and cell surfaces. Because of its strategic location and ability to store and protect growth factors, perlecan has been implicated in the control of tumor cell growth and metastatic behavior. To test the role of perlecan in malignancy, we generated several stably transfected clones of HT-1080, a human fibrosarcoma cell line, harboring a perlecan cDNA in the antisense orientation. Surprisingly, clones with a reduced synthesis of perlecan mRNA and protein core grew faster, formed larger colonies in semisolid agar, and induced faster formation of s.c. tumors in nude mice than the wild-type cells. Their growth properties in vitro were independent of exogenous basic fibroblast growth factor. Reduction of perlecan expression was associated with three distinct properties typical of tumor cells with a more aggressive phenotype: enhanced migration through 8-microm-pore filter, increased invasion in Matrigel-coated filters, and heightened adhesiveness to type IV collagen substrata. These results thus provide the first evidence that perlecan may inhibit the growth and invasiveness of fibrosarcoma cells in a basic fibroblast growth factor-independent pathway and raise the possibility that perlecan may prevent the infiltration of host tissues in mesenchymal neoplasms.     

Rac1 and extracellularly regulated kinase activation are sufficient for E1A-dependent cooperative transformation of primary epithelial cells, but progression can only be modulated by E1A or Rac1

Vibrissa connections play a decisive role in setting the somatotopic coordinates in the trigeminal sensory system. Although previous studies have examined the development of peripheral patterning, certain questions are still in dispute, for example, the way vibrissa connections are structured, and the relationship between periphery and central organization, and ganglion cell organization. In order to fill the blanks left by previous studies, the extension of ganglionic branches and the formation of vibrissa connections were reexamined by using fluorescent carbocyanin dyes, DiI and DiA, during embryonic days 10 to 14 in mouse. Whole-mount preparations satisfactorily demonstrated the ganglionic fiber system, which allowed detailed analysis at both macroscopic and microscopic levels. We show here that the differential fasciculation of follicular nerves is the critical process for organizing vibrissa connections which modulate the initially extending fiber pattern. Follicular nerves developed by fine fibers arising from initially ordered root fascicles, so as to connect with vibrissa rudiments that developed on the facial prominences. During differential fasciculation, ganglion cells were segregated into distinct groups by the vibrissa connections, whereas central fiber terminals did not yet develop specific structures in the nuclear region. In the primary order of the trigeminal sensory system, vibrissa connections in the periphery were organized before those of the central structure. These results indicate that trigeminal ganglion cells have a critical binomial function in order to transfer the somatotopic relations among vibrissa rudiments into the topographic coordinates of the central system.     

Interleukin-1-induced nuclear factor-kappaB-IkappaBalpha autoregulatory feedback loop in hepatocytes. A role for protein kinase calpha in post-transcriptional regulation of ikappabalpha resynthesis

We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy.     

An analysis of the role of the target membrane on the Gp64-induced fusion pore

Influenza hemagglutinin (HA) and GP64 of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus induce strikingly different initial fusion pores when mediating fusion between host cells that express these fusion proteins and target cells (Plonsky and Zimmerberg, 1996; Spruce et al., 1989, 1991; Zimmerberg et al., 1994). However, in these experiments, variations in host and target membranes confounded the analysis of the role that major components of the fusion reaction play in determining initial pore characteristics. To determine the contribution of the target cell plasma membrane to the fusion pore phenotype, we studied GP64-induced fusion of stably transfected cells (Sf9(Op1D)) to either red blood cells (RBCs) or Sf9 cells. Initial fusion pores in Sf9(Op1D)/RBC and Sf9(Op1D)/Sf9 cell pairs exhibited the same conductance, and pore flickering was not observed in either combination of cells. This indicates that the target cell determines neither the size nor the reversibility of the initial pore. However, the target cell does influence the kinetics of pore formation. The waiting time between triggering and pore appearance was shorter for Sf9(Op1D)/RBC fusion than for Sf9(Op1D)/Sf9 pairs. No correlation between pore waiting time and conductance was found. This argues against a molecular model that assumes aggregation of the pore wall from a nonfixed number of components as the rate-limiting step in GP64 fusion pore formation.     

S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells

S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.     

Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation

The receptor tyrosine kinase Kit plays critical roles in hematopoiesis, gametogenesis and melanogenesis. In mast cells, Kit receptor activation mediates several cellular responses including cell proliferation and suppression of apoptosis induced by growth factor deprivation and gamma-irradiation. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules. We have investigated the role of phosphatidylinositol 3'-kinase (PI 3-kinase) and Src kinases in Kit-mediated cell proliferation and suppression of apoptosis induced both by factor deprivation and irradiation in bone marrow-derived mast cells (BMMC). Analysis of Kit-/- BMMC expressing mutant Kit receptors and the use of pharmacological inhibitors revealed that both signaling pathways contribute to these Kit-mediated responses and that elimination of both pathways abolishes them. We demonstrate that the PI 3-kinase and Src kinase signaling pathways converge to activate Rac1 and JNK. Analysis of BMMC expressing wild-type and dominant-negative mutant forms of Rac1 and JNK revealed that the Rac1/JNK pathway is critical for Kit ligand (KL)-induced proliferation of mast cells but not for suppression of apoptosis. In addition, KL was shown to inhibit sustained activation of JNK induced by gamma-irradiation and concomitant irradiation-induced apoptosis.     

Fc receptor-mediated phagocytosis requires CDC42 and Rac1

At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.     

A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation

The hydrophilic bile salt ursodeoxycholic acid (UDCA) protects against the membrane-damaging effects associated with hydrophobic bile acids. This study was undertaken to (a) determine if UDCA inhibits apoptosis from deoxycholic acid (DCA), as well as from ethanol, TGF-beta1, Fas ligand, and okadaic acid; and to (b) determine whether mitochondrial membrane perturbation is modulated by UDCA. DCA induced significant hepatocyte apoptosis in vivo and in isolated hepatocytes determined by terminal transferase-mediated dUTP-digoxigenin nick end-labeling assay and nuclear staining, respectively (P < 0.001). Apoptosis in isolated rat hepatocytes increased 12-fold after incubation with 0.5% ethanol (P < 0.001). HuH-7 cells exhibited increased apoptosis with 1 nM TGF-beta1 (P < 0. 001) or DCA at >/= 100 microM (P < 0.001), as did Hep G2 cells after incubation with anti-Fas antibody (P < 0.001). Finally, incubation with okadaic acid induced significant apoptosis in HuH-7, Saos-2, Cos-7, and HeLa cells. Coadministration of UDCA with each of the apoptosis-inducing agents was associated with a 50-100% inhibition of apoptotic changes (P < 0.001) in all the cell types. Also, UDCA reduced the mitochondrial membrane permeability transition (MPT) in isolated mitochondria associated with both DCA and phenylarsine oxide by > 40 and 50%, respectively (P < 0.001). FACS(R) analysis revealed that the apoptosis-inducing agents decreased the mitochondrial transmembrane potential and increased reactive oxygen species production (P < 0.05). Coadministration of UDCA was associated with significant prevention of mitochondrial membrane alterations in all cell types. The results suggest that UDCA plays a central role in modulating the apoptotic threshold in both hepatocytes and nonliver cells, and inhibition of MPT is at least one pathway by which UDCA protects against apoptosis.     

A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membrane

Caveolin is a 22-kDa membrane protein found associated with a coat material decorating the inner membrane surface of caveolae. A remarkable feature of this protein is its ability to migrate from caveolae directly to the endoplasmic reticulum (ER) when membrane cholesterol is oxidized. We now present evidence caveolin is involved in transporting newly synthesized cholesterol from the ER directly to caveolae. MA104 cells and normal human fibroblasts transported new cholesterol to caveolae with a half-time of approximately 10 min. The cholesterol then rapidly flowed from caveolae to non-caveolae membrane. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Treatment of cells with 10 microg/ml progesterone blocked cholesterol movement from ER to caveolae. Simultaneously, caveolin accumulated in the lumen of the ER, suggesting cholesterol transport is linked to caveolin movement. Caveolae fractions from cells expressing caveolin were enriched in cholesterol 3-4-fold, while the same fractions from cells lacking caveolin were not enriched. Cholesterol transport to the cell surface was nearly 4 times more rapid in cells expressing caveolin than in matched cells lacking caveolin.     

Role of the TRAF binding site and NF-kappaB activation in Epstein-Barr virus latent membrane protein 1-induced cell gene expression

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-kappaB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-kappaB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IkappaBalpha blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-kappaB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.     

A role for RNA helicase A in post-transcriptional regulation of HIV type 1

Retroviruses must bypass the tight coupling of splicing and nuclear export of mRNA in their replication cycle because unspliced genomic RNA and incompletely spliced mRNA must be exported to the cytoplasm for packaging or translation. This process is mediated by a cis-acting constitutive transport element (CTE) for simple retroviruses and by the trans-acting viral protein Rev in concert with its response element (RRE) for complex retroviruses (e.g., HIV). Recently, we identified RNA helicase A (RHA) as a potential cellular cofactor for CTE. Here, we report that RHA also plays a role in Rev/RRE-mediated gene expression and HIV replication. RHA binds weakly to HIV-1 RRE independently of Rev. Overexpression of RHA, but not of an RHA mutant lacking helicase activity, increased both Rev/RRE- and CTE-dependent gene expression and the levels of unspliced HIV mRNA. Microinjection of antibodies to RHA into nuclei dramatically inhibited both CTE- and Rev-dependent gene expression in human cells. Exogenous RHA cDNA, but not the mutant RHA, rescued this inhibition. We propose that RHA is required to release both CTE- and RRE-containing mRNA from spliceosomes before completion of splicing, thus freeing them for nuclear export.     

Physiological role of Ca2+-permeable nonselective cation channel in endothelin-1-induced contraction of rabbit aorta

The alpha-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 degrees C and 55 degrees C in the presence and absence of cobalt, with an inverse relationship between alpha-amylase production and growth rate. Highest alpha-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h-1), at 40 degrees C in the absence of cobalt, while at the highest growth rate (0.35 h-1), at 55 degrees C in the presence of cobalt, alpha-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 micrograms maltose (mg biomass)-1 h-1. The pattern and levels of alpha-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.