Steady-state RNA Levels Research Articles

Inhibition of type I collagen gene expression in normal and systemic sclerosis fibroblasts by a specific inhibitor of geranylgeranyl transferase I.

To examine the effects of specific inhibition of geranylgeranyl transferase I on the expression of types I and III collagen genes in normal and systemic sclerosis (SSc) dermal fibroblasts in vitro. Fibroblasts from 2 normal subjects and 4 SSc patients were incubated with 2-10 microM of GGTI-298, a specific geranylgeranyl transferase inhibitor. Type I collagen and fibronectin production were determined by enzyme-linked immunosorbent assay. Steady-state messenger RNA (mRNA) levels for alpha1(I), alpha2(I), and alpha1(III) collagens and fibronectin were assessed by Northern hybridization, and the transcription of the alpha1(I) collagen gene was examined by transient transfections with a reporter construct containing -5.3 kb of the gene. GGTI-298 caused a dose-dependent inhibition of type I collagen production and a reduction in the steady-state levels of alpha1(I), alpha2(I), and alpha1(III) mRNA in normal and SSc cells. A 60-70% inhibition of type I collagen production and a 70-80% reduction in the mRNA levels for alpha1(I), alpha2(I), and alpha1(III) were observed at 10 microM GGTI-298. In contrast, the expression of fibronectin, cyclooxygenase 1, and GAPDH was not affected. The effects on alpha1(I) collagen mRNA resulted from a profound reduction in transcription of the alpha1(I) collagen gene promoter. GGTI-298 did not affect cellular viability or morphology. These results demonstrate that specific inhibition of geranylgeranyl prenylation causes a potent and selective inhibition of expression of the genes encoding types I and III collagens, without affecting cellular viability. The findings indicate that inhibition of geranylgeranyl prenylation should be further studied as a potential therapeutic approach for SSc and other fibrosing diseases.

Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression.

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.

Genetic evidence for a multi-subunit complex in the O-methyltransferase steps of coenzyme Q biosynthesis

Coq3 O-methyltransferase carries out both O-methylation steps in coenzyme Q (ubiquinone) biosynthesis. The degree to which Coq3 O-methyltransferase activity and expression are dependent on the other seven COQ gene products has been investigated. A panel of yeast mutant strains harboring null mutations in each of the genes required for coenzyme Q biosynthesis ( COQ1– COQ8) have been prepared. Mitochondria have been isolated from each member of the yeast coq mutant collection, from the wild-type parental strains and from respiratory deficient mutants harboring deletions in ATP2 or COR1 genes. These latter strains constitute Q-replete, respiratory deficient controls. Each of these mitochondrial preparations has been analyzed for COQ3-encoded O-methyltransferase activity and steady state levels of Coq3 polypeptide. The findings indicate that the presence of the other COQ gene products is required to observe normal levels of O-methyltransferase activity and the Coq3 polypeptide. However, COQ3 steady state RNA levels are not decreased in any of the coq mutants, relative to either wild-type or respiratory deficient control strains, suggesting either a decreased rate of translation or a decreased stability of the Coq3 polypeptide. These data are consistent with the involvement of the Coq polypeptides (or the Q-intermediates formed by the Coq polypeptides) in a multi-subunit complex. It is our hypothesis that a deficiency in any one of the COQ gene products results in a defective complex in which the Coq3 polypeptide is rendered unstable.

Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E 2 (PGE 2). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE 2 accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1α and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-β 2 and TGF-β 1 message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE 2 production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

Expression of betaig-h3 by human bronchial smooth muscle cells: localization To the extracellular matrix and nucleus.

Bronchial smooth muscle cells play a central role in normal lung physiology by controlling airway tone. In addition, airway smooth muscle hyperplasia and hypertrophy are important factors in the pathophysiology of asthma. In this study, expression of betaig-h3, a recently identified component of the extracellular matrix (ECM), was investigated in primary human bronchial smooth muscle (HBSM) cells. Northern blot analysis demonstrated that treatment of cultured HBSM cells with transforming growth factor-beta1 resulted in a 4- to 5-fold increase in the steady-state level of betaig-h3 messenger RNA. Western blot analysis of secreted proteins using monospecific antibodies generated against peptide sequences found in the N- and C-terminal regions of the protein identified several isoforms having apparent mass of 70-74 kD. Immunohistochemical analysis of human lung localized betaig-h3 to the vascular and airway ECM, and particularly to the septal tips of alveolar ducts and alveoli, suggesting that it may have a morphogenetic role. Analysis of cultured HBSM cells identified betaig-h3 in both the ECM as well as the cytoplasm, and surprisingly also in the nucleus. These results demonstrate that betaig-h3 is produced by resident lung cells, is a component of lung ECM, and may play an important role in lung structure and function. The presence of this protein in nuclei suggests that it may have regulatory functions in addition to its role as a structural component of lung ECM.

Direct action of naturally occurring estrogen metabolites on human osteoblastic cells.

This article describes experiments that were performed to examine the direct action of estrogen metabolites on cultured human osteoblast cells. The human fetal osteoblastic cell line, hFOB/ER9, which expresses high levels of the estrogen receptor (ER) alpha, was used to examine the direct effects of 16alpha-hydroxyestrone (16alpha-OHE1) and 2-hydroxyestrone (2-OHE1) on osteoblast differentiation. The 16alpha-OHE1 caused a decrease in osteocalcin (OC) secretion to a maximum of 40% of control values (vehicle-treated cells) at 10(-7) M. Alkaline phosphatase (AP) activity was significantly induced at 10(-7) M 16alpha-OHE1 with greater than 500% of control at 10(-6) M 16alpha-OHE1. Finally, AP steady-state messenger RNA (mRNA) levels were increased within 24 h of 16alpha-OHE1 treatment. In contrast to 16alpha-OHE1, 2-OHE1 had no effects on the secretion of OC, AP activity, or AP gene expression. The 2-OHE1 also did not display any antiestrogen activity because treatment in combination with 17beta-estradiol (E2) and 16alpha-OHE1 had no significant effect on the reduction in OC secretion or induction of AP activity. Similar to E2, 16alpha-OHE1 stimulated the expression of an early response gene, a TGF-beta inducible early gene, designated TIEG, as early as 60 minutes after treatment, whereas treatment with 2-OHE1 displayed no effect. Support that the 16alpha-OHE1 regulation of these osteoblasts (OB) markers was mediated through the ER is shown by the fact that the estrogen antagonist ICI 182,780 abrogated these effects. These data suggest that is a potent estrogen agonist on human osteoblastic hOB/ER9 cells. In contrast, 2-OHE1 displayed no estrogenic or antiestrogenic activity in this human osteoblast cell model.

Superoxide enhances interleukin 1β–mediated transcription of the hepatocyte-inducible nitric oxide synthase gene

Background & Aims: Exposure to oxidative stress, as in states of shock, ischemia-reperfusion injury, or sepsis, commonly initiates a complex cellular cascade of interlocking redox modulatory systems that detoxify electrophiles. In interleukin 1β (IL-1β)-treated rat hepatocytes, we have previously demonstrated that inducible nitric oxide synthase (iNOS) protein expression, steady-state iNOS messenger RNA (mRNA) levels, and NO synthesis are increased by oxidative stress induced by superoxide. The effect of hepatocellular redox state upon iNOS gene transcription has not been previously studied. Methods: Using rat hepatocytes in primary culture, iNOS gene transcription was induced by IL-1β. Oxidative stress was mediated by 1,2,3-benzenetriol (BZT), an autocatalytic source of superoxide. Nuclear run-on assays and transient transfection assays using the rat hepatocyte iNOS full-length promoter and deletion constructs were designed to isolate a cis-acting regulatory element. Specificity was confirmed by site-directed mutagenesis. Gel shift analysis determined the presence of a corresponding trans-acting regulatory factor. Results: In IL-1β–treated cells, BZT increased iNOS gene transcription without altering mRNA half-life. An antioxidant-responsive element (ARE) was found in the iNOS promoter at base pair –1347, which conferred redox sensitivity. Gel shift analysis identified a corresponding nuclear protein capable of binding to ARE in IL-1β– and BZT-treated rat hepatocytes. Conclusions: An ARE in the rat hepatocyte iNOS promoter confers redox sensitivity and augments IL-1β–mediated iNOS gene and protein expression in the setting of superoxide treatment. GASTROENTEROLOGY 2000;118:608-618

The influence of antisense gene location on target gene suppression in the fission yeast Schizosaccharomyces pombe.

A fission yeast model was employed to investigate the influence of antisense gene location on the efficacy of antisense RNA-mediated target gene suppression. Fission yeast transformants were generated that contained the target lacZ gene at a fixed position and a single copy antisense lacZ gene integrated into various genomic locations, including the same locus as the target gene. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus compared with other genomic locations, indicating that target and antisense gene colocalization is not a critical factor for efficient antisense RNA-mediated gene expression in vivo. Instead, increased lacZ downregulation correlated with an increase in antisense dose, with the steady-state levels of antisense RNA being dependent on genomic position effects and transgene copy number.

Breast tumour cell-induced down-regulation of type I collagen mRNA in fibroblasts.

This study investigated the modulation of type I collagen gene expression in normal fibroblasts by breast tumour cells. Northern analysis of total RNA extracted from stages I, II and III breast tumour tissue revealed that collagen mRNA levels were elevated in stage I tumours compared to the adjacent normal breast tissues, whereas they were decreased in stages II and III breast tumours. This aberrant collagen gene expression was confirmed by non-radioactive RNA:RNA in situ hybridization analysis of 30 breast carcinomas which localized the production of type I collagen mRNA to the stromal fibroblasts within the vicinity of the tumour cells. In order to determine whether the tumour cells were directly responsible for this altered collagen production by the adjacent fibroblasts, breast tumour cell lines were co-cultured with normal fibroblasts for in vitro assessment of collagen and steady-state collagen RNA levels. Co-culture of tumour cells and normal fibroblasts in the same dish resulted in down-regulation of collagen mRNA and protein. Treatment of the fibroblasts with tumour-cell conditioned medium also resulted in decreased collagen protein levels but the mRNA levels, however, remained unaltered. These results suggested that the tumour cells either secrete a labile ‘factor’, or express a cell surface protein requiring direct contact with the fibroblasts, resulting in down-regulation of collagen gene expression. Modulation of the ECM is a common characteristic of invading tumour cells and usually involves increased production of collagenases by the tumour cells or stromal fibroblasts. This study showed that tumour cells were also able to modulate collagen mRNA production by stromal fibroblasts, which may facilitate tumour cell invasion and metastasis. © 1999 Cancer Research Campaign

A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer.

The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18-25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

Structure and regulation of the salivary gland secretion protein gene Sgs-1 of Drosophila melanogaster.

The Drosophila melanogaster gene Sgs-1 belongs to the secretion protein genes, which are coordinately expressed in salivary glands of third instar larvae. Earlier analysis had implied that Sgs-1 is located at the 25B2-3 puff. We cloned Sgs-1 from a YAC covering 25B2-3. Despite using a variety of vectors and Escherichia coli strains, subcloning from the YAC led to deletions within the Sgs-1 coding region. Analysis of clonable and unclonable sequences revealed that Sgs-1 mainly consists of 48-bp tandem repeats encoding a threonine-rich protein. The Sgs-1 inserts from single lambda clones are heterogeneous in length, indicating that repeats are eliminated. By analyzing the expression of Sgs-1/lacZ fusions in transgenic flies, cis-regulatory elements of Sgs-1 were mapped to lie within 1 kb upstream of the transcriptional start site. Band shift assays revealed binding sites for the transcription factor fork head (FKH) and the factor secretion enhancer binding protein 3 (SEBP3) at positions that are functionally relevant. FKH and SEBP3 have been shown previously to be involved in the regulation of Sgs-3 and Sgs-4. Comparison of the levels of steady state RNA and of the transcription rates for Sgs-1 and Sgs-1/lacZ reporter genes indicates that Sgs-1 RNA is 100-fold more stable than Sgs-1/lacZ RNA. This has implications for the model of how Sgs transcripts accumulate in late third instar larvae.

Transient or sustained transcriptional activation of the genes encoding rat adrenomedullary catecholamine biosynthetic enzymes by different durations of immobilization stress

The impact of stress on the transcription of rat adrenal tyrosine hydroxylase and dopamine β-hydroxylase genes was examined. Nuclear run-on assays revealed that repeated immobilization stress elicited marked (about threefold) increases in the relative rates of transcription, being sustained for at least one day. Parallel changes in the steady-state messenger RNA levels for tyrosine hydroxylase and dopamine β-hydroxylase were also observed. A single episode of stress triggered similar enhancements in their relative transcription rates. Depending on the duration of the stress signal, the effect on gene transcription varied in its persistence. After very short (5 min) immobilization, there was a marked transient rise in the transcription of both genes, without an accumulation of the corresponding mRNAs. Longer episodes of stress (30 min) increased the relative rate of tyrosine hydroxylase transcription for hours, causing elevations in the steady-state messenger RNA levels. In contrast, although dopamine β-hydroxylase transcription was elevated to a similar extent by 30-min immobilization stress, the effect was transient and not reflected in significant accumulation of its messenger RNA. The results of our studies emphasize that the stress-evoked increases in the expression of the genes encoding adrenomedullary catecholamine biosynthetic enzymes involve transcriptional activation. Depending on the duration and reiteration of the stress signal, different transcriptional mechanisms may be employed.

Biallelic transcription of Igf2 and H19 in individual cells suggests a post-transcriptional contribution to genomic imprinting

The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3,4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.

Tumor necrosis factor-alpha mediates lipopolysaccharide-induced macrophage inflammatory protein-2 release from alveolar epithelial cells. Autoregulation in host defense.

Our recent studies have demonstrated that in response to lipopolysaccharide (LPS) challenge, alveolar epithelial cells produced tumor necrosis factor (TNF)-alpha, an early response cytokine in the inflammatory process. To investigate whether LPS-induced TNF-alpha release is related to other inflammatory mediators from the same cell type, we examined effects of LPS stimulation on macrophage inflammatory protein (MIP)-2 production by alveolar epithelial cells, and then examined the relationship between TNF-alpha and MIP-2 production. LPS stimulation induced a dose- and time-dependent release of MIP-2. The steady-state messenger RNA level of MIP-2 was significantly increased, with the MIP-2 protein localized within alveolar epithelial cells, as determined by confocal microscopy. The LPS-induced MIP-2 production is regulated at both the transcriptional and post-transcriptional levels. TNF-alpha also induced MIP-2 production from alveolar epithelial cells. Preincubation with an antisense oligonucleotide against TNF-alpha inhibited LPS-induced TNF-alpha in a dose-dependent and sequence-specific manner. The same antisense also inhibited MIP-2 production. The inhibitory effects were highly correlated. Polyclonal and monoclonal antibodies against TNF-alpha also attenuated LPS-induced MIP-2. These results suggest that LPS-induced MIP-2 release from alveolar epithelial cells may be mediated in part by TNF-alpha from the same cell type. This autoregulatory mechanism may amplify LPS-induced signals involved in host defense as well as in acute inflammatory reactions.

Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis.

Osteoporosis is a serious complication of systemic glucocorticoid use. However, while glucocorticoids increase bone resorption in vitro and in vivo, the mechanism(s) of this effect are at present unclear. Recent studies have identified the osteoprotegerin (OPG) ligand (OPG-L) as the final effector of osteoclastogenesis, an action that is opposed by the soluble neutralizing receptor, OPG. Thus, we assessed glucocorticoid regulation of OPG and OPG-L in various human osteoblastic lineage cells using Northern analysis, RT-PCR, and ELISA. Dexamethasone inhibited constitutive OPG messenger RNA (mRNA) steady-state levels by 70-90% in primary (MS) and immortalized stromal cells (hMS), primary trabecular osteoblasts (hOB), immortalized fetal osteoblasts (hFOB), and osteosarcoma cells (MG-63). In hFOB cells, dexamethasone inhibited constitutive OPG mRNA steady-state levels in a dose- and time-dependent fashion by 90%, and also suppressed cytokine-stimulated OPG mRNA steady-state levels. Dexamethasone-induced inhibition of OPG mRNA levels was not affected by the protein synthesis inhibitor, cycloheximide, and was shown to be due to inhibition of OPG gene transcription using a nuclear run-on assay. Moreover, dexamethasone also dose dependently (10(-10) M-10(-7) M) inhibited constitutive OPG protein concentrations in the conditioned medium of hFOB cells from 2.59 +/- 0.02 ng/ml (control) to 0.30 +/- 0.01 ng/ml (88% inhibition; P < 0.001 by ANOVA). Concurrently, dexamethasone stimulated OPG-L mRNA steady-state levels in MS and hFOB cells by 2- and 4-fold, respectively. Treatment of murine marrow cultures with conditioned medium harvested from dexamethasone-treated MG-63 cells increased tartrate-resistant acid phosphatase (TRAP) activity by 54% (P < 0.005) compared with medium harvested from control-treated cells (in the presence of OPG-L and macrophage colony-stimulating factor). Moreover, dexamethasone (10(-8) M) promoted osteoclast formation in vitro, as assessed by a 2.5-fold increase of TRAP activity in cell lysates (P < 0.001) and the appearance of TRAP-positive multinucleated cells. Our data are thus consistent with the hypothesis that glucocorticoids promote osteoclastogenesis by inhibiting OPG and concurrently stimulating OPG-L production by osteoblastic lineage cells, thereby enhancing bone resorption.

Human S100A11 Exhibits Differential Steady-State RNA Levels in Various Tissues and a Distinct Subcellular Localization

In order to analyze the steady-state RNA levels of S100A11 in different tissues, a cDNA fragment of human S100A11 was isolated from a cDNA library. The obtained fragment was labeled and hybridized to RNA isolated from various tissues. The Northern blot analysis revealed that S100A11 RNA levels varied from high in placenta, through intermediate in heart, lung, kidney, and most muscle samples, to barely detectable in brain. An efficient purification method for recombinant S100A11 yielding high quantities was developed. Furthermore, to examine the subcellular localization of this protein, the human polypeptide S100A11 antibodies were raised in rabbit. S100A11 was found to have a localization distinct from other S100 proteins examined, and is mostly localized in the nucleus, with slight variations among different glioblastoma cell types.

Inter-organellar crosstalk in higher plants: impaired chloroplast development affects mitochondrial gene and transcript levels.

Co-ordination of gene expression between the three genomes present in plastids, mitochondria and nucleus is of crucial importance for plant cells. Previous studies revealed that in white leaves of the albostrians (Hordeum vulgare cv. Haisa) mutant, photosynthesis-related plastid and nuclear genes are expressed only at an extremely low level. The plastids of this mutant lack ribosomes, photosynthetic activity and have only rudimentary membrane systems. Here we report on the expression of mitochondrial genes in albostrians barley. Steady-state RNA levels of the mitochondrial genes encoding cytochrome oxidase or ATPase subunits, coxII, coxIII, atpA, atp6, atp9 and cob, were observed to be consistently elevated in the white leaves but not in roots. Investigation of mitochondrial DNA revealed an about three-fold enhanced mitochondrial gene copy number in white compared to green leaf cells, but no differential amplification of mitochondrial genes. Analysis of plants in which the white albostrians plastids were combined with a new nuclear background showed that the enhanced transcript levels were a consequence of the impaired plastids and not of the nuclear albostrians allele. Furthermore, plants bleached by the carotenoid biosynthesis inhibitor norflurazon also showed an enhanced mitochondrial transcript level. These findings allow the conclusion that lack of chloroplast activity in an otherwise fully differentiated leaf leads to an increase in mitochondrial gene copy number and an elevated level of mitochondrial transcripts. Our results indicate an influence of plastids on the genetic apparatus of mitochondria in leaves but not in roots.

Sugars modulate an unusual mode of control of the cell-wall invertase gene (Incw1) through its 3' untranslated region in a cell suspension culture of maize.

We show here that a cell-wall invertase encoded by the Incw1 gene is regulated at both the transcriptional and posttranscriptional levels by sugars in a heterotrophic cell suspension culture of maize. The Incw1 gene encoded two transcripts: Incw1-S (small) and Incw1-L (large); the size variation was attributable to different lengths in the 3' untranslated region. Both metabolizable and nonmetabolizable sugars induced Incw1-L RNA apparently by default. However, only the metabolizable sugars, sucrose and D-glucose, were associated with the increased steady-state abundance of Incw1-S RNA, the concomitant increased levels of INCW1 protein and enzyme activity, and the downstream metabolic repression of the sucrose synthase gene, Sh1. Conversely, nonmetabolizable sugars, including the two glucose analogs 3-O-methylglucose and 2-deoxyglucose, induced greater steady-state levels of the Incw1-L RNA, but this increase did not lead to either an increase in the levels of the INCW1 protein/enzyme activity or the repression of the Sh1 gene. We conclude that sugar sensing and the induction of the Incw1 gene is independent of the hexokinase pathway. More importantly, our results also suggest that the 3' untranslated region of the Incw1 gene acts as a regulatory sensor of carbon starvation and may constitute a link between sink metabolism and cellular translation in plants.

Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis.

Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7. 2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.

Regulation of sodium iodide symporter gene expression in FRTL-5 rat thyroid cells.

The sodium iodide symporter (NIS), first identified in FRTL-5 cells, plays a critical role in iodide transport in the thyroid gland and in the production of the iodine-containing thyroid hormones. The aim of our study was to examine the regulation of NIS RNA steady-state levels and protein expression as well as functional activity in FRTL-5 cells. FRTL-5 cells cycling in media containing thyrotropin (TSH) were incubated for 48 hours with dexamethasone (10(-8)-10(-5) M), triiodothyronine (T3; 10(-9)-10(-6) M), methimazole (100 microM), propylthiouracil (PTU; 100 microM), perchlorate (10 microM) and potassium iodide (40 microM). In other experiments, cells were treated for 48 hours with various cytokines including interleukin-6 (IL-6) (100 U/mL), interferon-gamma (IFN-gamma) (100 U/mL), tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml), IL-1alpha (100 U/mL), and IL-1beta (100 U/mL). Northern blot analysis using a 32P-labeled rat NIS-specific cDNA probe (nucleotides 1397-1937) revealed NIS mRNA as a single species of approximately 3 kb. When normalized for beta-actin mRNA signal intensities, NIS RNA steady-state levels in viable FRTL-5 cells were suppressed by approximately 80% after incubation with dexamethasone and T3 in a concentration-dependent manner. Iodide accumulation was decreased by up to 40% after incubation with dexamethasone and T3, respectively, in a concentration-dependent manner. Using a rabbit polyclonal rNIS-specific antibody, Western blot analysis of FRTL-5 cell membranes revealed a 60% and 70% suppression of NIS protein expression after treatment with T3 (0.1 microM) and dexamethasone (1 microM), respectively. In additon, NIS RNA steady-state levels were decreased by approximately 50% after treatment of monolayers with methimazole, PTU, and potassium iodide, respectively. Incubation with methimazole and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. Treatment of FRTL-5 cells with IL-6 and IL-1beta resulted in a 30% decrease of NIS RNA steady-state levels. IL-6 did not alter NIS functional activity, but IL-1beta suppressed iodide accumulation by approximately 25%. IFN-gamma and perchlorate failed to alter NIS RNA steady-state levels. In contrast to IFN-gamma that had no effect on iodide accumulation, perchlorate almost completely suppressed iodide accumulation. TNF-alpha and IL-1alpha failed to alter NIS RNA steady-state levels in higher passage numbers of FRTL-5 cells, whereas treatment with TNF-alpha and IL-1alpha of early passages of FRTL-5 cells (<20 cell passages) resulted in a 70% and 40% decrease of NIS RNA steady-state levels, respectively, and in a 20% suppression of NIS functional activity. In conclusion, our data suggest that various agents known to affect iodide transport are capable of differentially altering NIS gene expression and function in cultured thyroid cells. Suppression of NIS gene expression and function by certain cytokines may be responsible, at least in part, for the impaired radioiodine uptake by thyroid tissue in certain forms of thyroiditis.