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