Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

The role of polyamine catabolism in polyamine analogue-induced programmed cell death

N1-ethyl-N11-[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT/polyamine oxidase (PAO) pathway produces H2O2 as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H2O2 production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (≥50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO by N,N′-bis(2,3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced by N1-ethyl-N11-[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H2O2 production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Activation of human monocytes induces differential resistance to apoptosis with rapid down regulation of caspase-8/FLICE

Cells of the monocyte/macrophage lineage play a central role in both innate and acquired immunity of the host. However, the acquisition of functional competence and the ability to respond to a variety of activating or modulating signals require maturation and differentiation of circulating monocytes and entail alterations in both biochemical and phenotypic profiles of the cells. The process of activation also confers survival signals essential for the functional integrity of monocytes enabling the cells to remain viable in microenvironments of immune or inflammatory lesions that are rich in cytotoxic inflammatory mediators and reactive free-radical species. However, the molecular mechanisms of activation-induced survival signals in monocytes remain obscure. To define the mechanistic basis of activation-induced resistance to apoptosis in human monocytes at the molecular level, we evaluated the modulation of expression profiles of genes associated with the cellular apoptotic pathways upon activation and demonstrate the following: (i) activation results in selective resistance to apoptosis particularly to that induced by signaling via death receptors and DNA damage; (ii) concurrent with activation, the most apical protease in the death receptor pathway, caspase-8/FLICE is rapidly down-regulated at the mRNA level representing a novel regulatory mechanism; and (iii) activation of monocytes also leads to dramatic induction of the Bfl-1 gene, an anti apoptotic member of the Bcl-2 family. Our findings thus provide a potential mechanistic basis for the activation-induced resistance to apoptosis in human monocytes.

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.

Extracellular Calcium Regulates HeLa Cell Morphology during Adhesion to Gelatin: Role of Translocation and Phosphorylation of Cytosolic Phospholipase A2

Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA.

Differential Roles of Syntaxin 7 and Syntaxin 8 in Endosomal Trafficking

To understand molecular mechanisms that regulate the intricate and dynamic organization of the endosomal compartment, it is important to establish the morphology, molecular composition, and functions of the different organelles involved in endosomal trafficking. Syntaxins and vesicle-associated membrane protein (VAMP) families, also known as soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), have been implicated in mediating membrane fusion and may play a role in determining the specificity of vesicular trafficking. Although several SNAREs, including VAMP3/cellubrevin, VAMP8/endobrevin, syntaxin 13, and syntaxin 7, have been localized to the endosomal membranes, their precise localization, biochemical interactions, and function remain unclear. Furthermore, little is known about SNAREs involved in lysosomal trafficking. So far, only one SNARE, VAMP7, has been localized to late endosomes (LEs), where it is proposed to mediate trafficking of epidermal growth factor receptor to LEs and lysosomes. Here we characterize the localization and function of two additional endosomal syntaxins, syntaxins 7 and 8, and propose that they mediate distinct steps of endosomal protein trafficking. Both syntaxins are found in SNARE complexes that are dissociated by α-soluble NSF attachment protein and NSF. Syntaxin 7 is mainly localized to vacuolar early endosomes (EEs) and may be involved in protein trafficking from the plasma membrane to the EE as well as in homotypic fusion of endocytic organelles. In contrast, syntaxin 8 is likely to function in clathrin-independent vesicular transport and membrane fusion events necessary for protein transport from EEs to LEs.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Differential quantal release of histamine and 5-hydroxytryptamine from mast cells of vesicular monoamine transporter 2 knockout mice

The recent availability of mice lacking the neuronal form of the vesicular monoamine transporter 2 (VMAT2) affords the opportunity to study its roles in storage and release. Carbon fiber microelectrodes were used to measure individual secretory events of histamine and 5-hydroxytryptamine (5-HT) from VMAT2-expressing mast cells as a model system for quantal release. VMAT2 is indispensable for monoamine storage because mast cells from homozygous (VMAT2−/−) mice, while undergoing granule-cell fusion, do not release monoamines. Cells from heterozygous animals (VMAT2+/−) secrete lower amounts of monoamine per granule than cells from wild-type controls. Investigation of corelease of histamine and 5-HT from granules in VMAT2+/− cells revealed 5-HT quantal size was reduced more than that of histamine. Thus, although vesicular transport is the limiting factor determining quantal size of 5-HT and histamine release, intragranular association with the heparin matrix also plays a significant role.

Differential requirement of the cytoplasmic subregions of γc chain in T cell development and function

The common cytokine receptor γ chain (γc), a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. The cytoplasmic domain of γc consists of 85 aa, in which the carboxyl-terminal 48 aa are essential for its interaction with and activation of the Janus kinase, Jak3. Evidence has been provided that Jak3-independent signals might be transmitted via the residual membrane-proximal region; however, its role in vivo remains totally unknown. In the present study, we expressed mutant forms of γc, which lack either most of the cytoplasmic domain or only the membrane-distal Jak3-binding region, on a γc null background. We demonstrate that, unlike γc or Jak3 null mice, expression of the latter, but not the former mutant, restores T lymphopoiesis in vivo, accompanied by strong expression of Bcl-2. On the other hand, the in vitro functions of the restored T cells still remained impaired. These results not only reveal the hitherto unknown role of the γc membrane-proximal region, but also suggest the differential requirement of the cytoplasmic subregions of γc in T cell development and function.

Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells

Previous studies have demonstrated hematopoietic stem cell amplification in vitro after the activation of three cell-surface receptors: flt3/flk2, c-kit, and gp130. We now show flt3-ligand and Steel factor alone will stimulate >85% of c-kit+Sca-1+lin− adult mouse bone marrow cells to proliferate in single-cell serum-free cultures, but concomitant retention of their stem cell activity requires additional exposure to a ligand that will activate gp130. Moreover, this response is restricted to a narrow range of gp130-activating ligand concentrations, above and below which hematopoietic stem cell activity is lost. These findings indicate a unique contribution of gp130 signaling to the maintenance of hematopoietic stem cell function when these cells are stimulated to divide with additional differential effects dictated by the intensity of gp130 activation.

NPH4, a Conditional Modulator of Auxin-Dependent Differential Growth Responses in Arabidopsis1

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein.

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves1

We investigated the relationship between H2O2 metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H2O2 content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O2·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.

Arabidopsis Rho-Related GTPases: Differential Gene Expression in Pollen and Polar Localization in Fission Yeast1

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.