Search

Background: ATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy., Methods: Two oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors., Results: ATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2 Tyr105 , p-PKM2 Ser37 , E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response., Conclusions: Inhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo., (© 2023. The Author(s).)

The Thyroid Hormone (TH) activating enzyme, type 2 Deiodinase (D2), is functionally required to elevate the TH concentration during cancer progression to advanced stages. However, the mechanisms regulating D2 expression in cancer still remain poorly understood. Here, we show that the cell stress sensor and tumor suppressor p53 silences D2 expression, thereby lowering the intracellular THs availability. Conversely, even partial loss of p53 elevates D2/TH resulting in stimulation and increased fitness of tumor cells by boosting a significant transcriptional program leading to modulation of genes involved in DNA damage and repair and redox signaling. In vivo genetic deletion of D2 significantly reduces cancer progression and suggests that targeting THs may represent a general tool reducing invasiveness in p53-mutated neoplasms., (© 2023. The Author(s).)

Inside the adult CNS, oligodendrocyte progenitor cells (OPC S ) are able to proliferate, migrate and differentiate into mature oligodendrocytes (OLs) which are responsible for the production of myelin sheet and energy supply for neurons. Moreover, in demyelinating diseases, OPCs are recruited to the lesion areas where they undergo differentiation and myelin synthesis. Serotonin (5-hydroxytryptamine, 5-HT) is involved in OLs' development and myelination, but so far the molecular mechanisms involved or the effects of 5-HT on mitochondria function have not yet been well documented. Our data show that 5-HT inhibits migration and proliferation committing cells toward differentiation in an immortalized human oligodendrocyte precursor cell line, M03-13. Migration blockage is mediated by reactive oxygen species (ROS) generation since antioxidants, such as Vit C and Cu-Zn superoxide dismutase, prevent the inhibitory effects of 5-HT on cell migration. 5-HT inhibits OPC migration and proliferation and increases OL phenotypic markers myelin basic protein (MBP) and Olig-2 via protein kinase C (PKC) activation since the inhibitor of PKC, bis-indolyl-maleimide (BIM), counteracts 5-HT effects. NOX inhibitors as well, reverse the effects of 5-HT, indicating that 5-HT influences the maturation process of OPCs by NOX-dependent ROS production. Finally, 5-HT increases mitochondria function and antioxidant activity. The identification of the molecular mechanisms underlying the effects of 5-HT on maturation and energy metabolism of OPCs could pave the way for the development of new treatments for autoimmune demyelinating diseases such as Multiple Sclerosis where oligodendrocytes are the primary target of immune attack.

Background: Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as D-serine and D-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of D-amino acids are found. We recently demonstrated that Ddo, the D-aspartate degradation gene, is actively demethylated to ultimately reduce D-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate D-ser levels during brain development has been poorly investigated to date., Methods: We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based D-amino acids level analyses of genes controlling the mammalian brain levels of D-serine and D-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis., Results: We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar D-serine a few days after mouse birth. High amount of 5'-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process., Conclusion: The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of D-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.

We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

DNA methylation is often analyzed by reporting the average methylation degree of each cytosine. In this study, we used a single molecule methylation analysis in order to look at the methylation conformation of individual molecules. Using D-aspartate oxidase as a model gene, we performed an in-depth methylation analysis through the developmental stages of 3 different mouse tissues (brain, lung, and gut), where this gene undergoes opposite methylation destiny. This approach allowed us to track both methylation and demethylation processes at high resolution. The complexity of these dynamics was markedly simplified by introducing the concept of methylation classes (MCs), defined as the number of methylated cytosines per molecule, irrespective of their position. The MC concept smooths the stochasticity of the system, allowing a more deterministic description. In this framework, we also propose a mathematical model based on the Markov chain. This model aims to identify the transition probability of a molecule from one MC to another during methylation and demethylation processes. The results of our model suggest that: 1) both processes are ruled by a dominant class of phenomena, namely, the gain or loss of one methyl group at a time; and 2) the probability of a single CpG site becoming methylated or demethylated depends on the methylation status of the whole molecule at that time.

The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a "reverse" oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of "reverse" oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis.

Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene.

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2-protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2-protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The proliferation and differentiation of muscle precursor cells require myogenic regulatory factors and chromatin modifiers whose concerted action dynamically regulates access to DNA and allows reprogramming of cells towards terminal differentiation. Type 2 deiodinase (D2), the thyroid hormone (TH)-activating enzyme, is sharply upregulated during myoblast differentiation, whereas type 3 deiodinase (D3), the TH-inactivating enzyme, is downregulated. The molecular determinants controlling synchronized D2 and D3 expression in muscle differentiation are completely unknown. Here, we report that the histone H3 demethylating enzyme (LSD-1) is essential for transcriptional induction of D2 and repression of D3. LSD-1 relieves the repressive marks (H3-K9me2-3) on the Dio2 promoter and the activation marks (H3-K4me2-3) on the Dio3 promoter. LSD-1 silencing impairs the D2 surge in skeletal muscle differentiation while inducing D3 expression thereby leading to a global decrease in intracellular TH production. Furthermore, endogenous LSD-1 interacts with FoxO3a, and abrogation of FoxO3-DNA binding compromises the ability of LSD-1 to induce D2. Our data reveal a novel epigenetic control of reciprocal deiodinases expression and provide a molecular mechanism by which LSD-1, through the opposite regulation of D2 and D3 expression, acts as a molecular switch that dynamically finely tunes the cellular needs of active TH during myogenesis.

Glucose is an essential fuel for cell survival and its availability limits aberrant cellular proliferation. We have hypothesized that specific cancer mutations regulate metabolic response(s) to glucose deprivation (GD). By means of somatic knock-in cellular models, we have analyzed the response to glucose deprivation in cells carrying the frequent (delE746-A750)EGFR, (G13D)KRAS or (E545K)PIK3CA cancer alleles. We demonstrate that, in mammary epithelial cells, glucose has an essential antioxidant function and that these cells are very sensitive to GD. Conversely, isogenic cells carrying the (delE746-A750)EGFR or the (E545K)PIK3CA, but not the (G13D)KRAS allele, display high tolerance to GD by stimulating the expression of anti-oxidant genes (MnSOD and catalase). This adaptive transcriptional response is mediated by the activation of WNT/β-catenin and FOXO4 signalling. Our data highlights a new functional synergism between oncogenic EGFR and PIK3CA with WNT/β-catenin conferring high tolerance to oxidative stress generated by nutrient deprivation.

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

To find a common pathogenetic trait induced by polyQ-expanded proteins, we have used a conditional expression system in PC12 cells to tune the expression of these proteins and analyze the early and late consequences of their expression. We find that expression for 3 h of a polyQ-expanded protein stimulates cellular reactive oxygen species (ROS) levels and significantly reduces the mitochondrial electrochemical gradient. 24-36 h later, ROS induce DNA damage and activation of the checkpoint kinase, ATM. DNA damage signatures are reversible and persist as long as polyQ-expanded proteins are expressed. Transcription of neural and stress response genes is down-regulated in these cells. Selective inhibition of ATM or histone deacetylase rescues transcription and restores the expression of silenced genes. Eventually, after 1 week, the expression of polyQ-expanded protein also induces endoplasmic reticulum stress. As to the primary mechanism responsible for ROS generation, we find that polyQ-expanded proteins, including native Ataxin-2 and Huntingtin, are selectively sequestered in the lipid raft membrane compartment and interact with gp91, the membrane NADPH-oxidase subunit. Selective inhibition of NADPH oxidase or silencing of H-Ras signaling dissolves the aggregates and eliminates DNA damage. We suggest that targeting of the polyQ-expanded proteins to the lipid rafts activates the resident NADPH oxidase. This triggers a signal linking H-Ras, ROS, and ERK1/2 that maintains and propagates the ROS wave to the nucleus. This mechanism may represent the common pathogenetic signature of all polyQ-expanded proteins independently of the specific context or the function of the native wild type protein.

Menin, the nuclear protein encoded by the Multiple Endocrine Neoplasia type 1 (MEN1) gene, acts as a tumor suppressor. It interacts with a large number of proteins involved in chromatin modification, transcription, cell cycle checkpoint and DNA repair, though its exact function is not clear. We report that in human cells menin stimulates homology-directed (HD) DNA repair induced by the rare endonuclease I-SceI and it accumulates with Chk1 at the site of the double strand break. In addition, menin and Chk1 interact in vivo. Deletion of the first 228 amino acids of menin impairs the interaction with Chk1 and the ability to stimulate HD repair, suggesting that the complex menin-Chk1 on the damaged chromatin facilitates homologous recombination., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Aims: The aim of the present study was to determine the function and the role of the scaffold protein AKAP121, tethering cAMP dependent protein kinase A to the outer wall of mitochondria, in neonatal ventricular myocytes and the heart., Methods and Results: Competitive peptides displacing AKAP121 from mitochondria in the tissue and in the cells were used to investigate the role of AKAP121 in mitochondrial function, reactive oxygen species (ROS) generation, and cell survival. Displacement of AKAP121 from mitochondria by synthetic peptides triggers the death program in cardiomyocytes. Under pathological conditions in vivo, in a rat model of cardiac hypertrophy induced by ascending aorta banding, the levels of AKAP121 are significantly down-regulated. Disappearance of AKAP121 is associated with mitochondrial dysfunction, high oxidative stress, and apoptosis. In vivo delocalization of AKAP121 by competitive peptides replicates some of the molecular signatures induced by pressure overload: mitochondrial dysfunction, increased mitochondrial ROS, and apoptosis., Conclusion: These data suggest that AKAP121 regulates the response to stress in cardiomyocytes, and therefore AKAP121 downregulation might represent an important event contributing to the development of cardiac dysfunction.

Background: The most frequent mutation of Friedreich ataxia (FRDA) is the abnormal expansion of a GAA repeat located within the first intron of FXN gene. It is known that the length of GAA is directly correlated with disease severity. The effect of mutation is a severe reduction of mRNA. Recently, a link among aberrant CpG methylation, chromatin organisation and GAA repeat was proposed., Methods: In this study, using pyrosequencing technology, we have performed a quantitative analysis of the methylation status of five CpG sites located within the region upstream of GAA repeat, in 67 FRDA patients., Results: We confirm previous observation about differences in the methylation degree between FRDA individuals and controls. We showed a direct correlation between CpG methylation and triplet expansion size. Significant differences were found for each CpG tested (ANOVA p<0.001). These differences were largest for CpG1 and CpG2: 84.45% and 76.80%, respectively, in FRDA patients compared to 19.65% and 23.34% in the controls. Most importantly, we found a strong inverse correlation between CpG2 methylation degree and age of onset (Spearman's rho = -0.550, p<0.001)., Conclusion: Because epigenetic changes may cause or contribute to gene silencing, our data may have relevance for the therapeutic approach to FRDA. Since the analysis can be performed in peripheral blood leucocytes (PBL), evaluation of the methylation status of specific CpG sites in FRDA patients could be a convenient biomarker.

We have recently reported that a novel MBD2 interactor (MBDin) has the capacity to reactivate transcription from MBD2-repressed methylated promoters even in the absence of demethylation events. Here we show that another unrelated protein, TACC3, displays a similar activity on methylated genes. In addition the data reported here provide possible molecular mechanisms for the observed phenomenon. Immunoprecipitation experiments showed that MBD2/TACC3 form a complex in vivo with the histone acetyltransferase pCAF. MBD2 could also associate with HDAC2, a component of MeCP1 repression complex. However, we found that the complexes formed by MBD2 with TACC3/pCAF and with HDAC2 were mutually exclusive. Moreover, HAT enzymatic assays demonstrated that HAT activity associates with MBD2 in vivo and that such association significantly increased when TACC3 was over-expressed. Overall our findings suggest that TACC3 can be recruited by MBD2 on methylated promoters and is able to reactivate transcription possibly by favoring the formation of an HAT-containing MBD2 complex and, thus, switching the repression potential of MBD2 in activation even prior to eventual demethylation.

c-Jun is a member of the activator protein 1 family, and its interaction with different nuclear factors generates a wide spectrum of complexes that regulate transcription of different promoters. H ferritin promoter transcription is tightly dependent on nuclear factor Y (NFY). Ferritin transcription is activated by c-Jun, although the promoter does not contain a canonical binding site. NFY, on the other hand, does not bind c-Jun in vitro, whereas in vivo c-Jun is found in the complex containing NFY. Moreover, a c-Jun-GCN4 chimaeric construct containing only the transactivation domain of Jun and the basic-region leucine-zipper domain of GCN4 stimulates the H ferritin promoter. A synthetic GAL4 promoter and the cognate activator, the fusion protein NFY-GAL4, are potently activated by c-Jun. Titration of p300 by co-expressing E1A abolishes the stimulatory effect. Moreover, another p300-dependent promoter, the cAMP-response element, can be superactivated by c-Jun using the same mechanism. These data indicate that c-Jun, when activated or overexpressed, is recruited to the H ferritin promoter by p300, which links NFY, bound to DNA, to the complex. These results add a new level of complexity to transcriptional regulation by c-Jun, which can activate p300-dependent promoters without binding directly to the target DNA.

Mitogen-activated protein kinases (MAPK) play a central role in signal transduction by regulating many nuclear transcription factors involved in inflammatory, immune, and proliferative responses. The aim of this study was to investigate, in human pulmonary endothelial cells, the effects of synthetic glucocorticosteroids on activation of c-jun N-terminal kinases, extracellular signal-regulated kinases, and p38 subgroups of the MAPK family. Human microvascular endothelial cells from lung were stimulated for 2 h with either H(2)O(2) (2 mM), IL-1beta (10 ng/mL), or tumour necrosis factor-alpha (10 ng/mL). Under these conditions, a remarkable increase in the phosphorylation pattern of c-jun N-terminal kinases, extracellular signal-regulated kinases 1/2, and p38 was detected. Pretreatment for 12 h with dexamethasone (100 nM) was able to prevent phosphorylation-dependent MAPK activation in stimulated cells, without substantially affecting the expression levels of these enzymes. Our results suggest that inhibition of MAPK signaling pathways in human pulmonary endothelial cells may significantly contribute, by interfering with activation of several different transcription factors, to the antiinflammatory and immunosuppressive effects of glucocorticosteroids.

Thyroid transcription factor 1 (TTF1) is a nuclear homeodomain protein that binds to and activates the promoters of several thyroid-specific genes, including that of the thyroglobulin gene (pTg). These genes are also positively regulated by thyroid-stimulating hormone/cyclic AMP (cAMP)/protein kinase A (PKA) signaling. We asked whether PKA directly activates TTF1. We show that cAMP/PKA activates pTg and a synthetic target promoter carrying TTF1 binding site repeats in several cell types. Activation depends on TTF1. Phosphopeptide mapping indicates that TTF1 is constitutively phosphorylated at multiple sites, and that cAMP stimulated phosphorylation of one site, serine 337, in vivo. However, alanine substitution at this residue or at all sites of phosphorylation did not reduce PKA activation of pTg. Thus, PKA stimulates TTF1 transcriptional activity in an indirect manner, perhaps by recruiting to or removing from the target promoter another regulatory factor(s).

Multinodular goiter (MNG) is characterized by nodules of different size and function. Areas of increased function may emerge, appearing as single, or more frequently, multiple autonomously functioning thyroid nodules (AFTN). The molecular mechanism for the autonomous growth and function of these nodules has been related to mutations in the thyrotropin receptor (TSHR) that constitutively activate the adenylyl cyclase. We searched for mutations in a limited area of the TSHR gene, covering the major mutational hotspot, in 38 AFTNs found in 37 patients with MNGs. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction enzyme analysis of fine-needle aspiration biopsy (FNAB) samples to rapidly identify 4 of the more frequently occurring TSHR mutations: D619G, F631C, T632I and D633E. Mutations were identified in 5 nodules (1 D619G mutation and 4 T632I mutations). Subsequently, the entire transmembrane portion of the TSHR gene was sequenced in a random sample of 12 AFTN samples that were free of mutations by RT-PCR and restriction enzyme analysis. By direct sequencing we identified a new mutation, F666L, in the seventh transmembrane domain in a sample from 1 nodule. Analysis of FMA samples of AFTN is an effective approach to identify TSHR gene mutations because individual mutations may be associated with different growth and function in vitro, our approach might, allow correlation of a given mutation with the clinical behavior in vivo.

We report that the heterotrimeric transcription factor NFY or "CAAT-binding factor" binds the -60 region of the human H ferritin promoter, the B site. DNA binding analysis with specific antibodies demonstrates that NFY/B/C subunits tightly bind this site and that NFY/C subunit is masked in vivo by binding with other protein(s). NFY binds the co-activator p300. Specifically, the NFY/B subunit interacts with the central segment of p300 in vivo and in vitro. cAMP substantially increases the formation of the NFY.p300 complex. Taken together these data provide a general model of cAMP induction of non-CRE-containing promoters and suggest that the NFY-B.p300 complex is located at the 5' end of the promoter and the NFY-B.C. TFIIB on the 3' end toward the transcription start site.

Detection of systemic tumor dissemination in colon carcinoma patients might be important for selection of appropriate treatment modalities. It has been previously shown that Apolipoprotein A-I (Apo A-I) is expressed in human intestinal epithelial cells, and in some human colon carcinoma cell lines. We examined the expression of Apo A-I mRNA in 14 human primary colon carcinomas by Northern blot and/or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. An Apo A-I specific transcript was found in up to 70% of the colon carcinomas. We developed an RT-PCR assay for Apo A-I transcripts, to identify circulating carcinoma cells in the peripheral blood of colon cancer patients. The Apo A-I RT-PCR assay was optimized using limiting dilution of an Apo A-I positive cancer cell line mixed with peripheral blood from healthy donor. In this system, up to 10 colon carcinoma cells were detected in 5 ml of peripheral blood. We examined Apo A-I mRNA expression in peripheral blood samples from 4 healthy donors, 20 colon carcinoma patients, and 11 individuals with tumor disease other than colon cancer. No Apo A-I mRNA was detected in the healthy donors and in the patients without colon cancer. Two out of 10 patients with metastatic colon carcinoma were positive by this assay, whereas Apo A-I mRNA was not found in any of the blood samples from the 10 radically resected colon carcinoma patients. These data suggest that Apo A-I RT-PCR assay is a highly specific and sensitive assay, although a low number of advanced colon carcinoma patients was found to be positive.

Expression of mutated versions of the p53 gene deranged the differentiation program of thyroid cells and resulted in deregulated growth. Specifically, p53 mutants in several residues of the DNA-binding region induced thyrotropin (TSH) -independent growth and inhibition of the expression of thyroid-specific genes. The loss of the differentiated phenotype invariably correlated with the blockage of the expression of the genes coding for the thyroid transcriptional factors PAX-8 and TTF2. Conversely, thyroid cells transfected with a p53 gene mutated at codon 392, located outside the DNA-binding region, stimulated the expression of differentiation genes in the absence of the TSH, and induced TSH-independent growth. cAMP intracellular levels were higher in thyroid cells transfected with the p53 gene mutated at the 392 site than in the untransfected thyroid cells, but lower in the cells transfected with the other mutated p53 genes. Fra-1 and c-jun were induced by p53, resulting in increased AP-1 levels. The results of this study suggest that p53 exerts effects on cAMP transduction pathway in thyroid cells, which are exquisitely sensitive to cAMP.

Transcription of the H ferritin gene in vivo is stimulated by cAMP and repressed by the E1A oncoprotein. We report here the identification of the cis-element in the human promoter responsive to both cAMP- and E1A-mediated signals. This promoter region is included between positions -62 to -45 and binds a approximate 120-kDa transcription factor called Bbf. Bbf forms a complex in vivo with the coactivator molecules p300 and CBP. Recombinant E1A protein reduces the formation of these complexes. In vivo overexpression of p300 in HeLa cells reverses the E1A-mediated inhibition of the ferritin promoter transcription driven by Bbf. These data suggest the existence of a common mechanism for the cAMP activation and the E1A-mediated repression of H ferritin transcription.

v-erbA is the oncogenic form of the c-erbA proto-oncogene, which encodes the receptor for thyroid hormones. The expression of the v-erbA oncogene in thyroid differentiated cells, PC Cl 3, inhibits iodide uptake and thyrotropin-dependent growth, whereas it has no effect on the expression of the other thyroid specific markers, i.e. thyroglobulin, thyroperoxidase and thyrotropin receptor. The activity of transcription factor AP-1, evaluated by a specific DNA binding assay and by transcription of AP-induced promoter (TRE) is enhanced in PC v-erbA cells. v-erbA mutants in the DNA binding domain do not affect the iodide uptake of thyroid cells nor AP-1 activity. We suggest that this transcriptional activation mediates the selective effects of v-erbA on the expression of thyroid specific markers.

We analyzed the expression and relative distribution of mRNA for the regulatory subunits (RIalpha, RIIalpha, and RIIbeta) and of 150-kDa RIIbeta-anchor proteins for cyclic AMP (cAMP)-dependent protein kinase (PKA) into discrete brain regions. The subcellular distribution of both holoenzyme and free catalytic subunit was evaluated in the same CNS areas. In the neocortex and corpus striatum high levels of RIIbeta paralleled the presence of specific RII-anchoring proteins, high levels of membrane-bound PKA holoenzyme, and low levels of cytosolic free catalytic activity (C-PKA). Conversely, in brain areas showing low RIIbeta levels (cerebellum, hypothalamus, and brainstem) we found an absence of RII-anchoring proteins, low levels of membrane-bound holoenzyme PKA, and high levels of cytosolic dissociated C-PKA. Response to cAMP stimuli was specifically evaluated in the neocortex and cerebellum, prototypic areas of the two different patterns of PKA distribution. We found that cerebellar holoenzyme PKA was highly sensitive to cAMP-induced dissociation, without, however, a consistent translocation of C-PKA into the nucleus. In contrast, in the neocortex holoenzyme PKA was mainly in the undissociated state and poorly sensitive to cAMP. In nuclei of cortical cells cAMP stimulated the import of C-PKA and phosphorylation of cAMP-responsive element binding protein. Taken together, these data suggest that RIIbeta (whose distribution is graded throughout the CNS, reaching maximal expression in the neocortex) may represent the molecular cue of the differential nuclear response to cAMP in different brain areas, by controlling cAMP-induced holoenzyme PKA dissociation and nuclear accumulation of catalytic subunits.

Tissue factor is a transmembrane protein that activates the extrinsic coagulation pathway by binding factor VII. Endothelial cells, being in contact with circulating blood, do not normally express tissue factor. Here we provide evidence that oxygen free radicals induce tissue factor messenger RNA transcription and expression of tissue factor procoagulant activity in endothelial cells in culture. Isolated, perfused rabbit hearts exposed to exogenous oxygen free radicals also showed a marked increase in tissue factor activity within the coronary circulation. Furthermore, in ex vivo and in vivo hearts subjected to ischemia and reperfusion, a condition associated with a production of oxygen free radicals in large amounts, a marked increase in tissue factor activity occurred. This phenomenon could be abolished by oxygen radical scavengers. This increase in tissue factor activity during postischemic reperfusion was accompanied by a significant decrease in coronary flow, suggesting that increase in tissue factor activity with the consequent activation of the coagulation cascade might impair coronary flow during reperfusion and possibly contribute to the occurrence of reperfusion injury.

Background: A variable degree of smooth muscle cell (SMC) proliferation after balloon injury has been reported in previous rat studies. It is unknown whether balloon injury induces c-fos expression and whether it is related to the degree of vascular injury in vivo. Therefore, we tested the hypothesis that proportional increases in neointimal formation and c-fos expression might be present after different degrees of balloon dilation., Methods and Results: Angioplasty of the carotid artery was performed with a balloon catheter. Vascular injury was evaluated at 0, 0.5, 1.0, 1.5, and 2 atm (n = 6 for all). In 40 additional rats, total RNA dot blots were performed to assess the effect of various degrees of balloon injury on c-fos expression. SMC proliferation proportional to the increases of inflation pressure was found between 0 and 2 atm with neointimal areas of 0.002 +/- 0.002, 0.069 +/- 0.014, 0.128 +/- 0.043, 0.190 +/- 0.010, and 0.255 +/- 0.041 mm2, respectively. When the degree of SMC proliferation (neointima and neointima/media ratio) was plotted against balloon inflation pressure, a linear relation was observed (r = .733, P < .001 and r = .755, P < .001, respectively). An increase in c-fos expression proportional to the degree of injury was found 30 minutes after injury., Conclusions: Neointimal proliferation produced by balloon injury is related to balloon inflation pressure, supporting the concept of an SMC proliferative response proportional to the degree of injury. The increase in SMC proliferation is associated with a proportional increase in the early expression of the c-fos nuclear proto-oncogene.

The asyaloglycoprotein receptor is a dimer formed by two polypeptide chains abundantly expressed in the liver (RHL-1 and RHL-2). Using specific primers for the two polypeptide chains we measured, by semiquantitative reverse PCR (RT-PCR), the corresponding mRNAs in different rat tissues. We found that both RHL-1 and RHL-2 mRNAs are expressed in the liver, kidney, brain and thyroid. Under the same conditions we did not detect any specific mRNA in the spleen. In the brain these sequences are expressed along a posterior-anterior gradient. Cerebellum and brainstem display the highest expression of the brain RHL-1 and RHL-2 mRNAs. Tissues and regional distribution of this receptor suggest that other body districts besides liver may participate in the clearance of serum glycoproteins.

The expression of thyroglobulin and other thyroid-specific markers depends upon the activation of protein kinase A (PKA) by cyclic AMP. A rat thyroid cell line dedifferentiates when transformed with Ki-ras oncogene. The decrease in thyroglobulin gene expression parallels a reduction in the level of PKA nuclear catalytic subunit. We find that the activity of cAMP-responsive elements and thyroglobulin promoters is down-regulated in Ras-transformed cells. Transcription of a third cAMP-regulated gene, H-ferritin, is similarly reduced. cAMP-responsive element and H-ferritin expression were stimulated when intracellular cAMP levels were increased. Reactivation of the thyroglobulin promoter required depletion of PKC in addition to increased cAMP. We also find that v-Ras activation leads to a significant increase in membrane-bound PKC. These data support the idea that v-Ras via PKC inhibits the transmission of cAMP-PKA signals to the nucleus. We suggest that the thyroglobulin promoter is more sensitive than other cAMP-dependent promoters to reduced nuclear levels of PKA catalytic subunit.

Hyperfunctioning thyroid adenomas are clonal neoplasms with the intrinsic capacity of growing and differentiate independently of thyroid-stimulating hormone (TSH). We analysed the mRNA encoding thyrotropin receptor of 11 adenomas obtained by fine needle aspiration biopsy (FNAB) and found 7 mutants all located in three aminoacids clustered in the sixth transmembrane domain of the receptor. These mutations were somatic and specifically present in the tumour tissue. DNA sequence revealed that 80 to 90% of the mutations can be rapidly screened and identified by restriction enzyme analysis of the amplified cDNA obtained from the FNABs. The mutation Thr->Ile was introduced in the wild type receptor and expressed in mouse fibroblasts. These cells constitutively activate the transcription of a reporter gene under the control of cyclic AMP responsive element.

Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.

We have studied the expression of cell polarity in hybrids between two rat thyroid epithelial cells: FRT and FRTL-5. FRT cells are polarized but do not express tissue-specific properties, FRTL-5 are unpolarized and express many thyroid-specific genes. A and express many thyroid-specific genes. A pool of 170 hybrid clones and five independent clones were characterized. The chromosome complement was that expected from 1:1 fusion of the parental cells. No chromosome loss was observed for several generations. All hybrids were polarized as judged from: (1) morphology, (2) transepithelial resistance, (3) preferential secretion of several proteins either through the apical (e.g. thyroglobulin) or through the basolateral pole, and (4) basolateral trapping of iodide. On the other hand, the expression of thyroid-specific markers: thyroglobulin synthesis and secretion, trapping of iodide, thyrotropin-dependent growth and expression of specific membrane antigens, were greatly reduced or inhibited in the pool and in the isolated clones. We also found that reduction of thyroglobulin synthesis was correlated with the loss of activity of the trans-acting factor TgTF1. We conclude that cell polarity, a property of FRT cells, is dominant in the hybrids whereas thyroid differentiation is recessive.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.

A series of overlapping recombinant clones, which cover the alpha 2 (type I) collagen gene, have been isolated by stepwise screening of two libraries of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned cDNA containing alpha 2 collagen DNA sequences as hybridization probe. The other clones were obtained by a sequence of screenings using defined fragments of the successive genomic clones as hybridization probes. Several types of experiments indicated that the DNA of these clones are truly overlapping and span 55 kilobase pairs of contiguous DNA sequences in the chicken genome. Sequence analysis of small DNA segments of some of these clones confirm that they contain coding sequences which specify alpha 2 collagen. Electron microscopic analysis of hybrids between type I alpha 2 collagen mRNA and the overlapping genomic clones indicates that the chicken alpha 2 collagen gene has a length of at least 37 kilobases, about 7.4 times longer than the corresponding translatable cytoplasmic mRNA. The coding information for alpha 2 collagen is distributed in more than 50 coding sequences which are interrupted by intervening sequences of various sizes. The structure of the gene implies that the conversion of precursor RNA to mature mRNA for alpha 2 collagen includes at least 50 splicing events.

Sixty-four unrelated healthy subjects were studied for the detection of a DNA polymorphism at the 5' end of the insulin gene. No significant difference between the groups was found in blood glucose values at fasting and after an oral glucose load. A significant association was found between fasting (P less than 0.05) and after load plasma C-peptide levels (P less than 0.01) and the presence of a 1.6 Kb insertion at the 5' end of the insulin gene. A gene dose-dependent effect was noted, class 3/3 individuals having the lowest after-load C-peptide concentration and class 1/3 an intermediate level (F for the linear trend: P = 0.007). This might suggest that insulin gene polymorphism affects insulin secretion in healthy individuals. In order to confirm this, a subgroup of six class 3/3 and eight class 1/1 individuals subsequently underwent a hyperglycaemic clamp. The tissue sensitivity to insulin was similar in the two groups but glucose-stimulated insulin secretion was markedly impaired in homozygotes for the class 3 allele. In this group, insulin secretion was, on average, only one-third of that in class 1/1 individuals (P less than 0.02). Similarly impaired in class 3/3 persons was the glucose + arginine-stimulated insulin secretion (P less than 0.05). We conclude that the polymorphism at the 5' end of the insulin gene is associated with variations in insulin secretion in healthy humans.

Purified pig thyroid chromatin has been transcribed in vitro with Escherichia coli RNA polymerase. The transcript, analyzed by DNA-RNA hybridization, shows two major kinetic components: 40% of the transcript is copied by repetitive sequences present 100 times per haploid genome; another 25% anneals to DNA with a rate constant Kh 10-4 M - S-1, typical of single-copy sequences. The transcript annealed at cot = 40 M - S to fractions of 2000-nucleotide DNA, when banded in neutral CsCl gradient only hybridizes to the heavy side of the main band. At cot = 3000 M - S, another hybridizing fraction appears on the light side of the main band of the gradient. The reassociation properties of these fractions show that the heavy DNA fraction is reiterated about 100 times per haploid genome, whereas the light DNA appears as a unique sequence, associated to small repetitive elements. The transcript, analyzed by formamide/sucrose gradient, shows two peaks with sedimentation coefficients of 10 S and 4 S, respectively. The 10-S RNA, hybridized to native 2000-nucleotide-length DNA, has a Kh of 10-4 M - S-1 and a cot1/2 of 10(3) M - S, typical of single-copy sequences.

The entire rat thyroglobulin mRNA sequence (about 8500 nucleotides) has been cloned in five recombinant plasmids containing overlapping cDNA inserts. The 3' end of the mRNA is precisely defined by the poly (A) tail found in the furthest 3' end clone. Evidence that most of the 5' end is cloned come from size considerations and from a primer extension experiment. At the 3' end of the mRNA only one long open reading frame is present in the sequence of 3018 nucleotides that has been established. In the deduced protein sequence we have localized two thyroxine-forming sites in a region containing a high concentration of tyrosine residues.

We have measured the synthesis of thyroglobulin in two differentiated cell lines, FRTL-5 and FRTL-424, and two transformed thyroid cell lines, FRA and 1-5G. The untransformed cells actively synthesized and exported thyroglobulin in the medium: however, the FRTL-5 cell line synthesized seven times less thyroglobulin than the FRTL-424 cell line, even though both cell types contained equal amounts of functional thyroglobulin mRNA. In contrast the transformed cells expressed extremely low levels of thyroglobulin mRNA, even though there was no detectable change in gene structure or copy number as determined by Southern blot analysis. On the basis of these data we conclude that (a) the different levels of thyroglobulin synthesis in the two untransformed cell lines are due to stable post-transcriptional alterations in the biosynthesis of thyroglobulin and (b) the transformation of thyroid cells results in a substantial reduction in thyroglobulin gene expression.