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Retinoblastoma
protein acts as Pax 8 transcriptional coactivator.
Miccadei
S, Provenzano
C, Mojzisek
M, Natali
PG, Civitareale
D.
Molecular Pathology Laboratory, Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy.
Control of cell proliferation and differentiation by the retinoblastoma
protein (pRb) depends on its interactions with
key cellular substrates. Available data indicate that pRb
and the transcription factor Pax 8 play a
crucial role in the differentiation of thyroid follicular cells. In this
study, we show that pRb takes part in the
complex assembled on the thyroperoxidase gene
promoter acting as a transcriptional coactivator
of Pax 8. Accordingly, pRb
interacts with and potentiates Pax 8 transcriptional activity. In addition, we show
that the downregulation of pRb
gene expression, in thyrocytes, through RNA
interference results in a reduction of the thyroperoxidase
gene promoter activity mediated by the Pax
8-binding site. In agreement with these results and with the ability of
the adenoviral protein E1A to bind pRb, we show
that E1A downregulates Pax
8 activity and that such inhibition requires the E1A-Rb interaction.
Furthermore, we show that the Pax 8/pRb synergy
plays a role on the sodium/iodide symporter
gene expression as well.
PMID: 16007137 [PubMed - indexed for MEDLINE]
Expression,
regulation, and function of paired-box gene 8 in the human placenta and
placental cancer cell lines.
Ferretti
E, Arturi
F, Mattei
T, Scipioni
A, Tell G, Tosi
E, Presta
I, Morisi
R, Lacroix
L, Gulino
A, Russo D,
Damante
G, Filetti
S.
Department of Clinical Science, University of Rome, La Sapienza,
Rome, Italy.
Pax proteins are transcriptional regulators
that control a variety of developmental decisions in vertebrates. During
development, the paired-box gene 8 (PAX8) is expressed in the thyroid,
kidney, and several areas of the central nervous system. It is also
expressed in the adult thyroid gland, in which it mediates TSH-induced
modulation of the expression of important genes, such as those encoding thyroglobulin, thyroperoxidase,
and the sodium/iodide symporter (NIS). Thus far, placental expression of PAX8 has been
described only in mice. In the present study, we show that PAX8 is also
expressed in the human placenta at term. In an in vitro model of
placental cancer, the JAR choriocarcinoma cell
line, human chorionic gonadotropin
(hCG) increased levels
of PAX8 mRNA and protein, and gel retardation assays indicated that the
up-regulation of PAX8 protein expression is associated with an increase
in its DNA-binding activity. The effects of hCG were mimicked by forskolin,
indicating that they are cAMP dependent. Levels
of mRNA for the Wilms' tumor 1 (WT1) and NIS genes were increased in JAR cells by hCG treatment, whereas overexpression of PAX8 increased only levels of WT1
mRNA. In cells transfected with PAX8-specific
small interfering RNA, the stimulatory effects of hCG on WT1 mRNA levels were abolished, but
hormonal enhancement of NIS mRNA levels was unchanged. These findings indicate
that, in JAR cells, hCG
activates a cAMP-dependent pathway that can
up-regulate WT1 expression through PAX8.
Glutathionylation of two cysteine
residues in paired domain regulates DNA binding activity of Pax-8.
Cao X, Kambe F,
Lu X, Kobayashi N,
Ohmori S,
Seo H.
Department of Endocrinology and Metabolism, Division of
Molecular and Cellular Adaptation, Research Institute of Environmental
Medicine, Nagoya University, Nagoya 464-8601, Japan. kambe@riem.nagoya-u.ac.jp
We reported that the first two cysteine
residues out of three present in paired domain (PD), a DNA-binding
domain, are responsible for redox regulation of
Pax-8 DNA binding activity. We show that glutathionylation
of these cysteines has a regulatory role in PD
binding. Wild-type PD and its mutants with substitution of cysteine to serine were synthesized and named CCC,
CSS, SCS, SSC, and SSS according to the positions of substituted cysteines. They were incubated in a buffer containing
various ratios of GSH/GSSG and subjected to gel shift assay. Binding of
CCC, CSS, and SCS was impaired with decreasing GSH/GSSG ratio, whereas
that of SSC and SSS was not affected. Because [3H]glutathione
was incorporated into CCC, CSS, and SCS, but not into SSC and SSS, the
binding impairment was ascribed to glutathionylation
of the redox-reactive cysteines.
This oxidative inactivation of PD binding was reversed by a reductant dithiothreitol
and by redox factor (Ref)-1 in vitro. To
explore the glutathionylation in cells, Chinese
hamster ovary cells overexpressing CSS and SCS
were labeled with [35S]cysteine
in the presence of cycloheximide. Immunoprecipitation with an antibody against PD
revealed that treatment of the cells with an oxidant diamide
induced the 35S incorporation into both mutants, suggesting the PD glutathionylation in cells. Since the two cysteine residues in PD are conserved in all Pax members, this novel posttranslational
modification of PD would provide a new insight into molecular basis for
modulation of Pax function.
PMID: 15888455 [PubMed - indexed for MEDLINE]
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