HOS1, a genetic locus involved in cold-responsive gene expression in arabidopsis

Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A, COR47, COR15A, KIN1, and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19 degrees C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10 degrees C or higher. Compared with the wild type, hos1-1 plants are l ess cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.     

Expression of an N-terminal fragment of COP1 confers a dominant-negative effect on light-regulated seedling development in Arabidopsis

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.     

Role of neurofibromin in modulation of expression of the tyrosinase-related protein 2 gene

Tyrosinase-related protein 2 (TRP-2)/DOPAchrome tautomerase is an enzyme involved in melanin biosynthesis and plays an important role in cytoprotection by preventing the production of a toxic melanin precursor, 5,6-dihydroxyindole. Neurofibromin is the protein product of a gene linked to neurofibromatosis type 1 (NF1), which is characterized by multiple neurofibromas and abnormalities in skin pigmentation. To explore the pathogenesis of NF1, we studied the role of neurofibromin in the regulation of TRP-2 gene expression. By means of transient cotransfection assays, we show that the expression of a reporter gene under the control of the TRP-2 gene promoter was increased by a neurofibromin-dependent signal through the 71-bp region (positions -415 to -345). A Lys-to-Glu substitution at position 1425 in neurofibromin abrogated this activating function. A dominant negative Ki-ras inhibitor mimics neurofibromin's function, and additively increases TRP-2 promoter activity when coexpressed with neurofibromin. Therefore, we suggest that neurofibromin is involved in the regulation of TRP-2 gene expression. Moreover, we found a single case of a glioblastoma multiforme that expresses TRP-2 mRNA but not tyrosinase mRNA, suggesting that TRP-2 may function in human neural tissues under certain conditions.     

Clustering of the nitrite reductase gene and a light-regulated gene with nitrate assimilation loci in Chlamydomonas reinhardtii

Degradation of extracellular matrix takes place in areas of cell-matrix contacts and is partly carried out by the action of matrix metalloproteinases (MMP). MMP-2 is a member of the MMP family that has been associated with breast-cancer metastasis. In the present study, we investigated the association of MMP-2 to the surface of breast-cancer cells and revealed an MMP-2-binding site that is expressed on sparsely plated cells and which is progressively lost as the cells approach confluence. Gelatin zymography, immunostaining and flow cytometry of MDA-MB-231 cells from sparse cultures demonstrated binding both of latent and of activated exogenous MMP-2, while little or no binding of MMP-2 was observed in confluent culture. Analysis of the expression of MTI-MMP, TIMP-2 and alpha(v) integrin, 3 proteins shown to play a role in cell-surface association of MMP-2, revealed enhanced levels of these proteins in confluent MDA-MB-231 cells. Thus, the reduced MMP-2 binding to confluent cells is not related to a deficiency in these MMP-2-binding proteins. Taken together, these studies suggest that MMP-2 binding to the surface of breast-cancer cells is regulated by cell-cell interactions and that tumor cells invading from the main tumor mass can up-regulate their MMP-2-binding capacity to acquire greater invasive capacity.     

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.     

The Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase is encoded by a light-regulated gene in Chlamydomonas reinhardtii

OBJECTIVES: Current surgical treatment for a glottic cancer with significant subglottic extension is a total laryngectomy. The objective of this study was to expand laryngeal conservation procedures by using a reconstructive technique that allows for the repair of hemicricolaryngectomy defects. STUDY DESIGN: After resection of the ipsilateral thyroid, cricoid, and arytenoid for advanced T3 glottic cancer, the laryngeal defect was reconstructed by means of an autotransplanted segment of trachea in four patients. The reconstruction consisted of a transferable patch that was constructed from a segment of revascularized cervical trachea. METHODS: During a 14-day period, a 4-cm segment of cervical trachea was wrapped by a free radial forearm fascial flap. In the second stage, the glottic cancer was removed and the cervical trachea was isolated on its fascial blood supply and transformed into a patch that was used to repair the extended hemilaryngectomy defect. Two different patch designs were used. Two patients underwent reconstruction with a patch augmented at the glottic level (group A); two patients underwent reconstruction without glottic augmentation on the patch (group B). Tracheal continuity was restored by an end-to-end reanastomosis. The postreconstruction morphology of the two patch designs was compared with the preoperative laryngeal morphology. RESULTS: The autotransplantation technique led to complete restoration of the subglottic airway lumen in all four patients. Although the anterior-posterior glottic diameter was reduced by 36% in group A patients and by 43.5% in group B patients, a sufficient glottic airway lumen was obtained. The glottic sphincteric function was restored in both groups. CONCLUSIONS: Tracheal autotransplantation may be used reliably to repair hemicricolaryngectomy defects. Augmentation of the patch at the level of the glottis is not essential for successful rehabilitation.     

Pollination-enhanced expression of a receptor-like protein kinase related gene in tobacco styles

We have isolated a putative serine/threonine receptor kinase gene with an expression pattern indicating that it may play a role in the stylar response to pollination. Differential display PCR was used to select tobacco mRNAs with increased accumulation following pollination. NTS16, a cDNA identified by this method, is homologous to a ca. 2.4 kb mRNA primarily expressed in pistil tissues. Levels of this mRNA increase during floral development and are further increased by pollination reaching maximal accumulation 12-18 hours after pollination and then declining. mRNA levels can also be increased by the application of ethylene to unpollinated flowers. A polypeptide encoded by the NTS 16 open reading frame has sequence similarity to the catalytic domain of several receptor protein kinases from plants including the S-receptor kinases implicated in the rejection of self-pollen in Brassica species and the Pto gene product of tomato which confers resistance to a bacterial pathogen.     

Cloning and characterization of AtRGP1. A reversibly autoglycosylated arabidopsis protein implicated in cell wall biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.     

Interleukin-1 alpha gene expression and localization of interleukin-1 alpha protein during tumor promotion

Treatment of the dorsal epidermis of SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and dose-dependent stimulation of interleukin-1 alpha (IL-1 alpha) gene expression. Levels of IL-1 alpha mRNA were elevated as early as 15 min and peaked at 3-4 h after a single application of TPA (2 micrograms or 10 micrograms). IL-1 alpha gene expression increased in epidermal tissue isolated from SENCAR mice at 1, 3, 6, 10, 16, and 22 wk after a single treatment with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and subsequent twice-weekly application of 2 micrograms TPA. IL-1 alpha-immunoreactive protein was specifically localized within suprabasal keratinocytes in cutaneous tissue isolated from mice treated with DMBA-TPA for 1-22 wk and in nonproliferating cells located within papilloma tissue isolated from SENCAR mice at 22 wk after initiation and promotion. Basal cells within hyperplastic epidermis did not produce IL-1 alpha-immunoreactive protein. DMBA treatment alone did not induce IL-1 alpha gene expression. Injection of IL-1 alpha-specific antibodies (50 micrograms) into SENCAR mice via the tail vein 2 h before treatment with TPA (2 micrograms or 10 micrograms) significantly (P < 0.05) inhibited the skin thickening usually observed 24 h after treatment with TPA. Autoradiography studies showed that injection of anti-IL-1 alpha antibodies inhibited incorporation of [methyl-3H]thymidine by keratinocytes within the epidermis and by cells within hair follicles. It also inhibited neutrophil infiltration into the dermis, which usually results from topical application of TPA. These data suggest that IL-1 alpha is a pivotal cytokine produced by specific subpopulations of epidermal keratinocytes and that IL-1 alpha primarily regulates the epidermal proliferative response of a distinctly separate population of keratinocytes after topical exposure of murine epidermis to TPA and secondarily modulates neutrophil migration into the dermis. Consequently, manipulation of IL-1 alpha may be a way to attenuate or abrogate the cutaneous response to TPA by altering keratinocyte proliferation, the resultant hyperplasia, and a portion of the inflammatory response characterized by dermal infiltration of neutrophils.     

Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression

The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.     

Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.     

New COP1-binding motifs involved in ER retrieval

Coatomer-mediated sorting of proteins is based on the physical interaction between coatomer (COP1) and targeting motifs found in the cytoplasmic domains of membrane proteins. For example, binding of COP1 to dilysine (KKXX) motifs induces specific retrieval of tagged proteins from the Golgi back to the endoplasmic reticulum (ER). Making use of the two-hybrid system, we characterized a new sequence (deltaL) which interacts specifically with the delta-COP subunit of the COP1 complex. Transfer of deltaL to the cytoplasmic domain of a reporter membrane protein resulted in its localization in the ER, in yeast and mammalian cells. This was due to continuous retrieval of tagged proteins from the Golgi back to the ER, in a manner similar to the ER retrieval of KKXX-tagged proteins. Extensive mutagenesis of deltaL identified an aromatic residue as a critical determinant of the interaction with COP1. Similar COP1-binding motifs containing an essential aromatic residue were identified in the cytoplasmic domain of an ER-resident protein, Sec71p, and in an ER retention motif previously characterized in the CD3epsilon chain of the T-cell receptor. These results emphasize the role of the COP1 complex in retrograde Golgi-to-ER transport and highlight its functional similarity with clathrin-adaptor complexes.     

Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 gene expression

Adhesion molecules such as VCAM-1 and ICAM-1 are increased in the central nervous system (CNS) during inflammatory responses and contribute to extravasation of leukocytes across the blood-brain barrier (BBB) and into CNS parenchyma. Astrocytes contribute to the structural integrity of the BBB and can be induced to express VCAM-1 and ICAM-1 in response to cytokines such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated the influence of IL-6 on astroglial adhesion molecule expression. IL-6, the soluble form of the IL-6R (sIL-6R), or both IL-6 plus sIL-6R, had no effect on VCAM-1 or ICAM-1 gene expression. Interestingly, the IL-6/sIL-6R complex inhibited TNF-alpha-induced VCAM-1 gene expression but did not affect TNF-alpha-induced ICAM-1 expression. The inhibitory effect of IL-6/sIL-6R complex was reversed by the inclusion of anti-IL-6R and gp130 Abs, demonstrating the specificity of the response. A highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide, which is designated H-IL-6, also inhibited TNF-alpha-induced VCAM-1 expression. sIL-6R alone was an effective inhibitor of TNF-alpha-induced VCAM-1 due to endogenous IL-6 production. These results indicate that the IL-6 system has an unexpected negative effect on adhesion molecule expression in glial cells and may function as an immunosuppressive cytokine within the CNS.