Cell Mol Life Sci. 2009;66:994-1009.
DNA repair 2008; 7:1155-67
Progress in DNA Damage Research, 2008
Neuroscience 2007;145:1213-21
Mut Research 2007; 614:3-15
Proc Natl Acad Sci USA. 2006; 103:16188-93
Mol Cell Biol 2006; 26:8722-8730

BioEssay 2003; 25:168-173
DNA Repair 2002; 1:59-75

Mol Cell Biol. 2004; 24:10670-80
J Invest Dermatol 2002 Feb;118(2):344-51
Mol Cel Biol 2000 Mar;20(5):1562-70
Exp Cell Res 1998 Aug 25;243(1):22-8
Proc Natl Acad Sci USA 1997 Apr 1;94(7):3116-21
Science 1997 Feb 14;275(5302):990-3 ..........Retracted
Hum Mol Genet 1994 Jun;3(6):963-7
Nature 1993 May 13;363(6425):182-5

Eur J Biochem 1992 Nov 15;210(1):365-73
FEBS Lett 1992 Apr 13;301(1):115-8
J Biol Chem 1989 Dec 25;264(36):21824-9
Proc Natl Acad Sci USA 1989 Oct;86(20):7838-42

Cell Mol Life Sci. 2009; 66:994-1009

DNA repair in mammalian cells : Nucleotide excision repair: variations on versatility.

Thierry Nouspikel
Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. It can be subdivided into several, differentially regulated, subpathways: global genome repair (GGR), transcription-coupled repair (TCR), and transcription domain-associated repair (DAR). This review begins with a brief overview of the numerous types of DNA lesions handled by NER, and proceeds to describe in detail the molecular mechanisms of NER. It then addresses heterogeneities in NER activity in physiological situations (e. g. in differentiated cells) and explores the underlying regulatory mechanism. It then reviews several inherited diseases associated with NER deficiencies: xeroderma pigmentosum, Cockayne syndrome, trichothiodystrophy, UV-sensitive syndrome. It concludes by discussing several currently unresolved issues, relating either to the cause of the above diseases or to the mechanistic details of the various NER subpathways and of their regulation.

PMID: 19153657

Progress in DNA Damage Research, 2008; Nova Science Publishers, Inc.

Differentiated Cells Play Favorites: Dissecting the Mechanisms of Discrimination in DNA Repair

Thierry Nouspikel
Nucleotide excision repair (NER) is one of the most important DNA repair systems, and certainly the most versatile: it can handle UV-induced lesions, bulky chemical adducts, protein-DNA adducts, intra-strand crosslinks, and it participates in the repair of inter-strand crosslinks. Hereditary deficiencies in NER result in several dreadful diseases, such as the highly cancer-prone xeroderma pigmentosum, and Cockayne syndrome, which is characterized by severe developmental and neurological defects.
Yet, despite its obvious importance, NER is strongly attenuated in many cell types; mostly in differentiated cells (neurons, macrophages, muscle fibres, etc.), but also in intermittent mitotic cells (e.g. B lymphocytes) and even in quiescent stem cells. A teleological explanation for this attenuation is that cells that do not divide may dispense with the burden of repairing the bulk of their genome, since they do not replicate it. This parsimonious strategy, however, demands that transcribed genes are still proficiently repaired for the cell to function normally. This is achieved by two subpathways of NER: transcription-coupled repair (TCR) and transcription domain-associated repair (DAR). DAR operates on both strands of active genes, whereas TCR enhances the repair rate of the transcribed strand by using the translocating RNA polymerase II as a lesion sensor.
Our recent work has shed some light on the mechanisms underlying NER attenuation in differentiated cells. We found that the ubiquitin-activating enzyme E1 is able to restore proficient NER in extracts from differentiated macrophages, suggesting that an NER enzyme must be activated by ubiquitination. This activation does not occur in differentiated cells, most likely because of a decrease in phosphorylation of the E1 enzyme.  The function of E1 is to activate ubiquitin and transfer it to a battery of E2 enzymes. It is likely that some of these E2s, such as the one used by NER, are unable to interact with the hypo-phosphorylated form of E1 that we have observed in macrophages.
The molecular details of TCR are still elusive, aside from the fact that a stalled RNA polymerase II recruits NER enzyme to the site of damage. By contrast, we have evidence that DAR is simply a concentration of NER enzymes in the nuclear sub-compartments in which transcription takes place. The remaining repair-proficient NER enzymes likely accumulate in these transcription factories, accounting for the persistence of NER in active genes, despite its attenuation at the global genomic level.

DNA Repair  2008; 7:1155-67

Nucleotide excision repair and neurological diseases.

Thierry Nouspikel
This review will examine the known and postulated relationships between nucleotide excision repair (NER) and neurological diseases. We will begin with a description of NER and its subpathways: global genomic repair (GGR), transcription-coupled repair (TCR) and transcription domain-associated repair (DAR). As far as they are known, the underlying molecular mechanisms will be discussed. We will only briefly touch on the possible contribution of NER to neurodegenerative diseases such as Alzheimer's, but concentrate on neurological symptoms in NER-deficient patients. These are mainly observed in two clinical entities, Xeroderma pigmentosum (XP) and Cockayne syndrome (CS), and we shall try to understand why and how a deficit in DNA repair may result in neurological dysfunctions. The links between NER and neurological disease are also discussed in contributions by Brooks and by Niedernhofer, in this volume.

PMID: 18456575

Proc Natl Acad Sci USA. 2006; 103:16188-93 

Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme.

Thierry Nouspikel and Philip C. Hanawalt
Global nucleotide excision repair is greatly attenuated in terminally differentiated mammalian cells. We observed this phenomenon in human neurons and in macrophages, noting that the transcription-coupled repair pathway remains functional and that there is no significant reduction in levels of excision repair enzymes. We have discovered that ubiquitin-activating enzyme E1 complements the repair deficiency in macrophage extracts, and although there is no reduction in the concentration of E1 upon differentiation, our results indicate a reduction in phosphorylation of E1. In preliminary studies, we have identified the basal transcription factor TFIIH as the potential target for ubiquitination. We suggest that this unusual type of regulation at the level of the E1 enzyme is likely to affect numerous cellular processes and may represent a strategy to coordinate multiple phenotypic changes upon differentiation by using E1 as a "master switch."

PMID: 17060614

Mol Cell Biol 2006; 26:8722-8730 

Transcription domain-associated repair in human cells.

Thierry Nouspikel, Nevila Hyka-Nouspikel, and Philip C. Hanawalt
Nucleotide excision repair (NER), which is arguably the most versatile DNA repair system, is strongly attenuated in human cells of the monocytic lineage when they differentiate into macrophages. Within active genes, however, both DNA strands continue to be proficiently repaired. The proficient repair of the nontranscribed strand cannot be explained by the dedicated subpathway of transcription-coupled repair (TCR), which is targeted to the transcribed strand in expressed genes. We now report that the previously termed differentiation-associated repair (DAR) depends upon transcription, but not simply upon RNA polymerase II (RNAPII) encountering a lesion: proficient repair of both DNA strands can occur in a part of a gene that the polymerase never reaches, and even if the translocation of RNAPII is blocked with transcription inhibitors. This suggests that DAR may be a subset of global NER, restricted to the subnuclear compartments or chromatin domains within which transcription occurs. Downregulation of selected NER genes with small interfering RNA has confirmed that DAR relies upon the same genes as global genome repair, rather than upon TCR-specific genes. Our findings support the general view that the genomic domains within which transcription is active are more accessible than the bulk of the genome to the recognition and repair of lesions through the global pathway and that TCR is superimposed upon that pathway of NER.

PMID: 17015469

Neuroscience 2007;145:1213-21

DNA repair in differentiated cells: Some new answers to old questions

Thierry Nouspikel
Terminally differentiated cells need never replicate their genomes and may therefore dispense with the daunting task of maintaining several repair systems to constantly scan their entire complement of DNA. Obviously, transcribed genes need to be repaired, so that cells can carry out their specialized functions, but dedicated mechanisms such as transcription-coupled repair and differentiation-associated repair can ensure the maintenance of those transcriptionally active domains. Many groups have studied DNA repair in differentiated cells, often with divergent results, possibly because there are distinct classes of differentiated cells, with unique properties. Thus neurons ought to last for a lifetime, whereas myocytes are backed by precursor cells, while white blood cells like macrophages are constantly being replaced. More importantly, different DNA repair systems can vary in their response to cellular differentiation, possibly depending on whether they can be coupled to transcription. Nucleotide excision repair (NER) is probably the most versatile DNA repair system and is coupled to transcription. NER was shown to be attenuated by differentiation in several cell types, including neurons. The attenuation occurs only at the global genome level, with transcribed genes still being efficiently repaired. We have determined that this attenuation results from the lack of ubiquitination of a NER factor, most likely owing to differences in phosphorylation of the ubiquitin-activating enzyme E1. Because there is only one E1 in human cells, it is likely that other metabolic pathways are similarly affected, depending on whether they rely on an E2 enzyme which is sensitive to the state of E1 phosphorylation.

PMID: 16920273

Mutation Research 2007; 614:3-15

Nucleotide excision repair phenotype of human acute myeloid leukemia cell lines at various stages of differentiation

Hsu Peihsin, Philip C. Hanawalt, and Thierry Nouspikel
In previous studies it was shown that nucleotide excision repair (NER) is strongly attenuated at the global genome level in terminally differentiated neuron-like cells. NER was measured in several human acute myeloid leukemia cell lines, before and after differentiation into macrophage-like cells. Repair of cisplatin intrastrand GTG crosslinks in differentiated cells was strongly attenuated. There were also some variations between repair levels in naive cells, but these were not correlated with the degree of differentiation. By contrast, the proficient repair of UV-induced (6-4)pyrimidine-pyrimidone photoproducts [(6-4)PPs] was not affected by differentiation. Although cyclobutane pyrimidine dimers (CPDs) were poorly repaired at the global genome level in all cell lines, differentiated or not, they were very efficiently removed from the transcribed strand of an active gene, indicating that transcription-coupled repair (TCR) is proficient in each cell line. CPDs were also removed from the non-transcribed strand of an active gene better than at the overall global genome level. This relatively efficient repair of the non-transcribed strand of active genes, when compared with global genomic repair (GGR), has been described previously in neuron-like cells and termed differentiation-associated repair (DAR). Here we show that it also can occur in actively growing cells that display poor GGR.

PMID: 16890248

Mol Cell Biol. 2004; 24:10670-80

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage

Fabrizio Thorel, Angelos Constantinou, Isabelle Dunand-Sauthier, Thierry Nouspikel, Philippe Lalle, Anja Raams, Nicolaas G. J. Jaspers, Wim Vermeulen, Mahmud K. K. Shivji, Richard D. Wood, and Stuart G. Clarkson
XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.

PMID: 15572672

BioEssays 2003; 25:168-173

When parsimony backfires: Neglecting DNA repair may doom neurons in Alzheimer's disease

Thierry Nouspikel and Philip C. Hanawalt
Taking advantage of the fact that they need not replicate their DNA, terminally differentiated neurons only repair their expressed genes and largely dispense with the burden of removing damage from most of their genome. However, they may pay a heavy price for this laxity if unforeseen circumstances, such as a pathological condition like Alzheimer's disease, cause them to re-enter the cell cycle. The lifetime accumulation of unrepaired lesions in the silent genes of neurons is likely to be significant and may result in aborting the mitotic process and triggering cell death if the cells attempt to express these dormant genes and resume DNA replication.

PMID: 12539243

DNA Repair 2002; 1:59-75

DNA repair in terminally differentiated cells

Thierry Nouspikel and Philip C. Hanawalt
Terminally differentiated cells do not replicate their genomic DNA, and could therefore dispense with the task of removing DNA damage from the non-essential bulk of their genome, as long as they are able to maintain the integrity of the genes that must be expressed. There is increasing experimental evidence that this is indeed the case, at least for some repair pathways such as nucleotide excision repair. In this review we examine a number of terminally differentiated cell systems in which it has been demonstrated that DNA repair is attenuated at the global genome level, but maintained in expressed genes. How these cells manage to repair transcribed genes is not yet fully elucidated, but there are indications that the transcription-coupled repair (TCR) pathway could maintain integrity of the transcribed strand in the active genes. We have observed in neurons that the non-transcribed strand of active genes is also well repaired, a phenomenon that we have named differentiation-associated repair (DAR). It is conceivable that DAR is necessary to maintain the integrity of the template strand that is needed by TCR to complete the repair of lesions in the transcribed strand of essential expressed genes with high fidelity.

PMID: 12509297

J Invest Dermatol 2002 Feb;118(2):344-51

The founding members of xeroderma pigmentosum group G produce XPG protein with severely impaired endonuclease activity.

Lalle P, Nouspikel T, Constantinou A, Thorel F, Clarkson SG.

Department of Genetics and Microbiology, Centre Medical Universitaire (CMU), Geneva, Switzerland.

Of the eight human genes implicated in xeroderma pigmentosum, defects in XPG produce some of the most clinically diverse symptoms. These range from mild freckling to severe skeletal and neurologic abnormalities characteristic of Cockayne syndrome. Mildly affected xeroderma pigmentosum group G patients have diminished XPG endonuclease activity in nucleotide excision repair, whereas severely affected xeroderma pigmentosum group G/Cockayne syndrome patients produce truncated XPG proteins that are unable to function in either nucleotide excision repair or the transcription-coupled repair of oxidative lesions. The first two xeroderma pigmentosum group G patients, XP2BI and XP3BR, were reported before the relationship between xeroderma pigmentosum group G and Cockayne syndrome was appreciated. Here we provide evidence that both patients produce truncated proteins from one XPG allele. From the second allele, XP2BI generates full-length XPG of 1186 amino acids containing a single L858P substitution that has reduced stability and greatly impaired endonuclease activity. In XP3BR, a single base deletion and alternative splicing at a rare noncanonical AT-AC intron produces a 1185 amino acid protein containing 44 internal non-XPG residues. This protein is stably expressed but it also has greatly impaired endonuclease activity. These four XPG products can thus account for the severe ultraviolet sensitivity of XP2BI and XP3BR fibroblasts. These cells, unlike those from xeroderma pigmentosum group G/Cockayne syndrome patients, are capable of limited transcription-coupled repair of oxidative lesions. Our results suggest that the L858P protein in XP2BI and the almost full-length XPG protein in XP3BR are responsible for this activity and for the absence of severe early onset Cockayne syndrome symptoms in these patients.

PMID: 15572672

Mol Cel Biol 2000 Mar;20(5):1562-70

Terminally Differentiated Human Neurons Repair Transcribed Genes but Display Attenuated Global DNA Repair and Modulation of Repair Gene Expression

Thierry Nouspikel and Philip C. Hanawalt

Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

Repair of UV-induced DNA lesions in terminally differentiated human hNT neurons was compared to that in their repair-proficient precursor NT2 cells. Global genome repair of (6-4)pyrimidine-pyrimidone photoproducts was significantly slower in hNT neurons than in the precursor cells, and repair of cyclobutane pyrimidine dimers (CPDs) was not detected in the hNT neurons. This deficiency in global genome repair did not appear to be due to denser chromatin structure in hNT neurons. By contrast, CPDs were removed efficiently from both strands of transcribed genes in hNT neurons, with the nontranscribed strand being repaired unexpectedly well. Correlated with these changes in repair during neuronal differentiation were modifications in the expression of several repair genes, in particular an up-regulation of the two structure-specific nucleases XPG and XPF/ERCC1. These results have implications for neuronal dysfunction and aging.

Exp Cell Res 1998 Aug 25;243(1):22-8

Complementation of transformed fibroblasts from patients with combined xeroderma pigmentosum-Cockayne syndrome.

Ellison AR, Nouspikel T, Jaspers NG, Clarkson SG, Gruenert DC

Laboratory Medicine and Stromatology, University of California San Francisco, San Francisco, California, 94143, USA.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40).The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene. Copyright 1998 Academic Press.

PMID: 9716445, UI: 98384333

Proc Natl Acad Sci U S A 1997 Apr 1;94(7):3116-21

A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: implications for a second XPG function.

Nouspikel T, Lalle P, Leadon SA, Cooper PK, Clarkson SG

Department of Genetics and Microbiology, University Medical Centre (CMU), Geneva 4, Switzerland.

Xeroderma pigmentosum (XP) patients have defects in nucleotide excision repair (NER), the versatile repair pathway that removes UV-induced damage and other bulky DNA adducts. Patients with Cockayne syndrome (CS), another rare sun-sensitive disorder, are specifically defective in the preferential removal of damage from the transcribed strand of active genes, a process known as transcription-coupled repair. These two disorders are usually clinically and genetically distinct, but complementation analyses have assigned a few CS patients to the rare XP groups B, D, or G. The XPG gene encodes a structure-specific endonuclease that nicks damaged DNA 3' to the lesion during NER. Here we show that three XPG/CS patients had mutations that would produce severely truncated XPG proteins. In contrast, two sibling XPG patients without CS are able to make full-length XPG, but with a missense mutation that inactivates its function in NER. These results suggest that XPG/CS mutations abolish interactions required for a second important XPG function and that it is the loss of this second function that leads to the CS clinical phenotype.

PMID: 9096355, UI: 97250499

Science 1997 Feb 14;275(5302):990-3

Defective transcription-coupled repair of oxidative base damage in Cockayne syndrome patients from XP group G.

Cooper PK, Nouspikel T, Clarkson SG, Leadon SA

Life Sciences Division, Building 934, Lawrence Berkeley National Laboratory, University of California, 1 Cyclotron Road, Berkeley, CA 94720, USA.

In normal human cells, damage due to ultraviolet light is preferentially removed from active genes by nucleotide excision repair (NER) in a transcription-coupled repair (TCR) process that requires the gene products defective in Cockayne syndrome (CS). Oxidative damage, including thymine glycols, is shown to be removed by TCR in cells from normal individuals and from xeroderma pigmentosum (XP)-A, XP-F, and XP-G patients who have NER defects but not from XP-G patients who have severe CS. Thus, TCR of oxidative damage requires an XPG function distinct from its NER endonuclease activity. These results raise the possibility that defective TCR of oxidative damage contributes to the developmental defects associated with CS.

This paper was retracted in 2005 because the experiments featured in Fig 1 and 3 could not be reproduced.

PMID: 9020084, UI: 97172564

Hum Mol Genet 1994 Jun;3(6):963-7

Mutations that disable the DNA repair gene XPG in a xeroderma pigmentosum group G patient.

Nouspikel T, Clarkson SG

Department of Genetics and Microbiology, Centre Medical Universitaire (CMU), Geneva, Switzerland.

The human XPG (ERCC5) gene encodes a large acidic protein that corrects the ultraviolet light sensitivity of cells from both xeroderma pigmentosum complementation group G and rodent ERCC group 5. Here we characterize five XPG sequence alterations and a minor splicing defect in XP-G patient XP125LO. Three of these changes are polymorphic variants whereas the remaining two, one in each XPG allele, inactivate complementation in vivo. These single point mutations provide formal proof that defects in XPG give rise to the group G form of xeroderma pigmentosum, and their locations suggest ways in which this may occur.

PMID: 7951246, UI: 95038755

Nature 1993 May 13;363(6425):182-5

Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2.

Scherly D, Nouspikel T, Corlet J, Ucla C, Bairoch A, Clarkson SG

Department of Genetics and Microbiology, University Medical Centre (CMU), Geneva, Switzerland.

Defects in human DNA repair proteins can give rise to the autosomal recessive disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), sometimes even together. Seven XP and three CS complementation groups have been identified that are thought to be due to mutations in genes from the nucleotide excision repair pathway. Here we isolate frog and human complementary DNAs that encode proteins resembling RAD2, a protein involved in this pathway in yeast. Alignment of these three polypeptides, together with two other RAD2 related proteins, reveals that their conserved sequences are largely confined to two regions. Expression of the human cDNA in vivo restores to normal the sensitivity to ultraviolet light and unscheduled DNA synthesis of lymphoblastoid cells from XP group G, but not CS group A. The XP-G correcting protein XPGC is generated from a messenger RNA of approximately 4 kilobases that is present in normal amounts in the XP-G cell line.

* Comment in: Nature 1993 May 13;363(6425):114-5

PMID: 8483504, UI: 93247645

Eur J Biochem 1992 Nov 15;210(1):365-73

Insulin signalling and regulation of glucokinase gene expression in cultured hepatocytes.

Nouspikel T, Iynedjian PB

Division of Clinical Biochemistry, University of Geneva School of Medicine, Switzerland.

In cultured rat hepatocytes, transcription of the glucokinase gene is turned on by insulin and turned off by glucagon/cAMP, the latter being the dominant effector system. It is thus possible that in the absence of hormones the gene is maintained in a repressed state by the basal level of cAMP and that insulin turns on transcription by relieving cAMP repression, for instance via activation of a cyclic-nucleotide phosphodiesterase. Three inhibitors of this class of enzymes were tested for their effect on the insulin-dependent induction of the glucokinase gene in hepatocytes. Isobutyl methylxanthine, the prototype inhibitor, abrogated the gene response to insulin, as shown by run-on transcription assay. Among the drugs investigated, Ly186126, a preferential inhibitor of type-III phosphodiesterase, proved the most potent in inhibiting insulin-induced accumulation of glucokinase mRNA. Type-III phosphodiesterase is inhibited by cGMP. Induction of glucokinase mRNA was prevented in hepatocytes challenged with insulin in presence of 8-bromoguanosine-3',5'-phosphate. These results are consistent with the involvement of type-III phosphodiesterase in transduction of the insulin signal to the glucokinase gene. However, we were unable to detect significant decreases in total cellular cAMP level or cAMP-dependent-protein-kinase ratio after the addition of insulin to hepatocytes. Many effects of glucagon are mediated via cAMP-dependent protein-kinase phosphorylation of regulatory proteins and, conversely, insulin effects are often accompanied by protein dephosphorylation. A specific inhibitor of protein phosphatases PP1 and PP2A, okadaic acid, was shown to abolish the transcriptional response of the glucokinase gene to insulin. Thus, interference of insulin with the cAMP signal transduction pathway at several steps may be a critical aspect of insulin action on hepatic glucokinase gene expression. In addition, insulin induction of glucokinase mRNA was suppressed by inhibitors of protein synthesis. The underlying mechanism was a severe inhibition of the transcriptional effect of insulin, rather than mRNA destabilization, as demonstrated by run-on transcription assays with nuclei from cycloheximide-treated or pactamycin-treated cells. Transcription of the glucokinase gene may therefore depend on de novo synthesis of the product of an early-response gene induced by insulin, or may require a short-lived trans-acting or accessory factor of transcription. Alternatively, insulin signalling may be compromised in hepatocytes by a mechanism indirectly related to the arrest of protein synthesis.

PMID: 1280218, UI: 93076810

FEBS Lett 1992 Apr 13;301(1):115-8

Unimpaired effect of insulin on glucokinase gene expression in hepatocytes challenged with amylin.

Nouspikel T, Gjinovci A, Li S, Iynedjian PB

Division of Clinical Biochemistry, University of Geneva School of Medicine, Switzerland.

Amylin appears to interfere with the action of insulin in muscle and possibly in liver. We have attempted to detect a direct antagonism between amylin and insulin in cultured rat hepatocytes. The stimulation of glucokinase gene expression was used as a marker of insulin action. Amylin proved ineffective in suppressing subsequent accumulation of glucokinase mRNA in response to maximal or submaximal doses of insulin. When applied to cells already induced by prior incubation with insulin alone, amylin failed to reverse induction, in contrast to the effectiveness of glucagon under the same conditions. Thus, amylin is not a physiological antagonist of insulin in the control of hepatic glucokinase gene expression.

PMID: 1451780, UI: 93083611

J Biol Chem 1989 Dec 25;264(36):21824-9

Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system.

Iynedjian PB, Jotterand D, Nouspikel T, Asfari M, Pilot PR

Institut de Biochimie Clinique, University of Geneva School of Medicine, Switzerland.

Primary cultures of rat hepatocytes were used to investigate the regulation of glucokinase gene expression by insulin and glucagon. Insulin added in physiological concentrations to the culture medium causes de novo induction of glucokinase mRNA. The induced plateau is reached 4 to 8 h after insulin addition, and the mRNA level remains high as long as insulin is present. Comparison of the potencies of insulin, proinsulin, and insulin-like growth factor I in this system indicates that induction by insulin is mediated via the insulin receptor. The magnitude of the insulin effect is independent of the extracellular glucose concentration. Run-on transcription assays with isolated nuclei show that the mRNA build up depends primarily on a specific stimulation of glucokinase gene transcription. Glucagon added to hepatocytes together with a supramaximal concentration of insulin prevents induction of glucokinase mRNA in a dose-dependent manner. The inhibitory effect of glucagon is mimicked by 8-(4-chlorophenylthio)-cAMP. The effect of this agent has also been tested in hepatocytes first induced for maximal glucokinase gene transcription by culture with insulin alone for 12 h. The transcriptional activity of the gene as measured by run-on assay was completely turned off within 30 min after addition of the cyclic nucleotide. Under these conditions, glucokinase mRNA decays rapidly, with an apparent half-life of 45 min. The mRNA degradation rate was similarly rapid after insulin withdrawal from induced cells. Thus, a cAMP-mediated repression mechanism is a key aspect in the regulation of glucokinase gene transcription in the hepatocyte. Insulin may act by relieving the gene from repression.

PMID: 2557341, UI: 90094361

Proc Natl Acad Sci U S A 1989 Oct;86(20):7838-42

Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.

Iynedjian PB, Pilot PR, Nouspikel T, Milburn JL, Quaade C, Hughes S, Ucla C, Newgard CB

Institut de Biochimie Clinique, University of Geneva School of Medicine, Switzerland.

Glucokinase, a key regulatory enzyme of glucose metabolism in mammals, provides an interesting model of tissue-specific gene expression. The single-copy gene is expressed principally in liver, where it gives rise to a 2.4-kilobase mRNA. The islets of Langerhans of the pancreas also contain glucokinase. Using a cDNA complementary to rat liver glucokinase mRNA, we show that normal pancreatic islets and tumoral islet cells contain a glucokinase mRNA species approximately 400 nucleotides longer than hepatic mRNA. Hybridization with synthetic oligonucleotides and primer-extension analysis show that the liver and islet glucokinase mRNAs differ in the 5' region. Glucokinase mRNA is absent from the livers of fasted rats and is strongly induced within hours by an oral glucose load. In contrast, islet glucokinase mRNA is expressed at a constant level during the fasting-refeeding cycle. The level of glucokinase protein in islets measured by immunoblotting is unaffected by fasting and refeeding, whereas a 3-fold increase in the amount of enzyme occurs in liver during the transition from fasting to refeeding. From these data, we conclude (i) that alternative splicing and/or the use of distinct tissue-specific promoters generate structurally distinct mRNA species in liver and islets of Langerhans and (ii) that tissue-specific transcription mechanisms result in inducible expression of the glucokinase gene in liver but not in islets during the fasting-refeeding transition.

PMID: 2682629, UI: 90046691