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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2001 as doi:10.1096/fj.00-0556fje. |
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,2
* Unidad de Hepatología y Terapia Génica, Departamento de Medicina Interna, Facultad de Medicina, Universidad de Navarra. 31008, Pamplona, Spain;
Departamento de Bioquímica y Biología Molecular, Universidad de Valencia. 46100, Burjassot, València, Spain;
Servicio de Hepatología, Hospital Clínico y Provincial. 08036, Barcelona, Spain; and
Center for Liver Disease Research and Division of Gastrointestinal Liver Diseases, Department of Medicine, University of Southern California School of Medicine, Los Angeles, California 90033, USA
3Correspondence: Departamento de Medicina Interna, Edificio Los Castaños, Facultad de Medicina, Universidad de Navarra, 31008, Pamplona, Spain. E-mail: jmmato{at}unav.es
SPECIFIC AIMS
We have studied the molecular mechanisms and mediators behind the induction of methionine adenosyltransferase 2 A (MAT2A) gene expression in the regenerating rat liver after partial hepatectomy. The involvement of hepatocyte growth factor (HGF) and cellular S-adenosylmethionine (AdoMet) levels in the regulation of MAT2A expression are evaluated in a model of cultured rat hepatocytes.
PRINCIPAL FINDINGS
1. Acetylation of histone H4 associated with MAT2A
promoter is tissue specific and enhanced in the remaining liver after
partial hepatectomy (PH)
In mammals, MAT2A is expressed in all cells of the
organism with the exception of the mature and quiescent hepatocyte.
Chromatin immunoprecipitation experiments using an antibody specific to
hyperacetylated histone H4 revealed enhanced acetylation of histone H4
associated with MAT2A promoter in an expressing tissue such
as kidney, whereas the opposite situation was observed in the liver. As
previously reported, MAT2A expression was induced in the
hepatic parenchymal cell shortly after PH. Activation of
MAT2A transcription was accompanied by time-dependent
enhancement in the acetylation status of histone H4 associated with its
promoter, as evidenced by chromatin immunoprecipitation assays.
2. HGF induces the hyperacetylation of histone H4 associated with
MAT2A promoter and MAT2A expression in
cultured rat hepatocytes
HGF is a key growth factor in the induction of hepatocyte
proliferation and one of the main stimuli leading to the rapid changes
in gene expression after PH. We studied its effect on MAT2A
expression in a model of cultured rat hepatocytes. In this experimental
system, we have observed that HGF (50 ng/ml, for 1 h) induces the
hyperacetylation of histone (H4) associated with MAT2A
promoter (Fig. 1
). This effect was blocked by the tyrosine kinase inhibitor genistein
(10 µg/ml) (Fig. 1)
. As would be expected from the effect of HGF on
MAT2A promoter-associated histones, transcription of
MAT2A was stimulated by this growth factor in a dose- and
time-dependent fashion. The induction of MAT2A transcription
by HGF was also impaired in the presence of genistein. HGF activated
the transcription of a reporter gene (luciferase) under the control of
MAT2A 5' region in transient transfection experiments
performed in cultured hepatocytes.
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3. AdoMet modulates HGF-induced MAT2A gene
expression and DNA synthesis in cultured rat hepatocytes
In isolated rat hepatocytes, we had previously shown that the
expression of MAT2A was progressively induced with time in
culture, whereas that of the liver-specific gene MAT1A was
dramatically reduced, probably reflecting the degree of
dedifferentiation of cultured hepatocytes. The addition of AdoMet to
the culture medium prevented such changes in MAT1A and
MAT2A expression. It was therefore important to know whether
MAT2A induction by HGF could be modulated by AdoMet.
Hepatocytes were preincubated with increasing concentrations of AdoMet
for 30 min and treated with HGF (50 ng/ml for 3 h). As shown in
Fig. 2A
, AdoMet addition resulted in the dose-dependent inhibition
of MAT2A expression by HGF. AdoMet effect may be related to
its conversion into 5'-methylthio-adenosine (MTA), a metabolite of
AdoMet in the polyamine biosynthetic pathway. Pretreatment of
hepatocytes with 500 µM of MTA effectively blocked the induction of
MAT2A expression by HGF, whereas the expression of
MAT1A was not affected (Fig. 2B
). It has been
reported that AdoMet or MTA administration to rats after PH results in
the impairment of DNA synthesis in the liver parenchymal cell. We have
now tested the effect of AdoMet on HGF-induced DNA synthesis in
cultured rat hepatocytes. In agreement with the in vivo
observations in rats after PH, AdoMet was able to partially
inhibit HGF-stimulated DNA synthesis (Fig. 2C
).
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CONCLUSIONS
Methionine adenosyltransferase (MAT) catalyzes the formation of AdoMet, a key metabolite central to most cellular transmethylation reactions and a precursor of polyamine biosynthesis. In the adult and quiescent hepatocyte, AdoMet is synthesized by MAT I/III, the product of MAT1A gene. When hepatocytes proliferate, however, as occurs during liver regeneration, malignant transformation, or the fetal period, transcription of MAT2A is activated resulting in the expression of MAT II, the form of MAT normally expressed outside the liver. Evidence has been reported showing that this switch in MAT gene expression provides the cell with a proliferative advantage, which may stem from the distinct regulatory and kinetic properties of MAT I/III and MAT II that influence intracellular AdoMet levels. However, nothing has been known about the mechanisms that govern MAT2A expression during the physiological proliferative response of the hepatocyte.
It is widely accepted that chromatin structure plays a crucial role in the regulation of gene expression in eukaryotes. The interplay of remodeling complexes and covalent histone modifications seems to be essential for the access of DNA binding factors. Histone acetylation is one of such covalent modifications that have been linked to transcriptional activity. We show that histones (H4) associated with MAT2A promoter are hyperacetylated in a tissue were the gene is expressed, such as kidney, but are hypoacetylated in the liver. When the hepatocyte proliferates, as in an experimental model of liver regeneration after PH, the acetylation status of histones (H4) associated with MAT2A promoter is markedly increased. These in vivo observations suggest that such changes in chromatin at the level of MAT2A promoter may play a role in transcriptional activation of this gene.
To identify the factors that could mediate the activation of MAT2A expression in the regenerating liver, we turned to an experimental system of isolated rat hepatocytes. HGF is responsible for many of the hepatocellular responses after PH, including the induction of early responsive genes and hepatocyte proliferation. Our observations in primary cultured hepatic cells have identified MAT2A as a novel target for HGF. Furthermore, MAT2A expression in response to HGF was preceded by the hyperacetylation of histones (H4) associated with its promoter. It has been proposed that the level of selective histone acetylation may depend on signal transduction pathways, but little is known about the possible signal cascades for which histone acetyltransferases (HATs) and/or deacetylases are the end points. It has been shown that steroid hormones and vitamins A and D are able to induce hyperacetylation of histones at promoters of target genes through the recruitment of p300/CBP HAT activity. Together with the recently reported ability of epidermal growth factor to promote the phosphorylation and acetylation of histone H3 associated with c-fos promoter, our present observations are the first report of such an effect for a tyrosine kinase-activating growth factor such as HGF. So far, our findings support the more general view that localized changes in chromatin structure induced by extracellular signals can be considered a common event in the dynamic regulation of gene expression. Whether HGF also promotes histone H4 phosphorylation or histone H3 modifications (phosphorylation/acetylation) remains to be determined.
The process of liver regeneration involves many complex
mechanisms that are not completely understood. A major area of research
in this field is the identification of the mechanisms that modulate
hepatocyte responsiveness to HGF at the onset of the proliferative
response and the signals that determine the termination of liver
regeneration. Together with growth factors and cytokines, changes in
key metabolite levels may contribute to the orchestration of the
regenerative process. We would like to propose that AdoMet and/or MTA
could be one of these key metabolites. Hepatic levels of both molecules
are dramatically reduced shortly after PH, when HGF levels rise and
MAT2A expression is activated. As intracellular AdoMet and
MTA concentrations subsequently recover to normal levels, the
hepatocyte would be rendered refractory to HGF at least regarding the
induction of MAT2A and DNA synthesis, as we observed in
cultured hepatocytes. Although the detailed mechanisms behind AdoMet
inhibition of MAT2A induction are not completely known, a
methylation reaction seems not to be involved, since MTA mimicked this
effect and is not a methyl donor compound. Our observations supporting
this novel hypothesis are summarized in Fig. 3
and allow us to suggest that fluctuations in the hepatic concentrations
of AdoMet and/or MTA could be part of the priming events and
terminating signals that modulate the liver regenerative
process.
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FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0556fjev1; to cite this
article, use FASEB J. (March 5, 2001)
10.1096/fj.00-0556fjev1 
2 Both authors made equal contribution to this
work.
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