124 research outputs found
Differential p38-dependent signalling in response to cellular stress and mitogenic stimulation in fibroblasts
p38 MAP kinase is known to be activated by cellular stress finally leading to cell cycle arrest or apoptosis. Furthermore, a tumour suppressor role of p38 MAPK has been proposed. In contrast, a requirement of p38 for proliferation has also been described. To clarify this paradox, we investigated stress- and mitogen-induced p38 signalling in the same cell type using fibroblasts. We demonstrate that - in the same cell line - p38 is activated by mitogens or cellular stress, but p38-dependent signalling is different. Exposure to cellular stress, such as anisomycin, leads to a strong and persistent p38 activation independent of GTPases. As a result, MK2 and downstream the transcription factor CREB are phosphorylated. In contrast, mitogenic stimulation results in a weaker and transient p38 activation, which upstream involves small GTPases and is required for cyclin D1 induction. Consequently, the retinoblastoma protein is phosphorylated and allows G1/S transition. Our data suggest a dual role of p38 and indicate that the level and/or duration of p38 activation determines the cellular response, i.e either proliferation or cell cycle arrest
Characterization of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Mutations in Familial Isolated Pituitary Adenoma Families
Familial isolated pituitary adenoma (FIPA) is an autosomal dominant condition with variable genetic background and incomplete penetrance. Germline mutations of the aryl hydrocarbon receptor interacting protein (AIP) gene have been reported in 15-40% of FIPA patients. Limited data are available on the functional consequences of the mutations or regarding the regulation of the AIP gene. We describe a large cohort of FIPA families and characterize missense and silent mutations using minigene constructs, luciferase and beta-galactosidase assays, as well as in silico predictions. Patients with AIP mutations had a lower mean age at diagnosis (23.6+/-11.2 years) than AIP mutation-negative patients (40.4+/-14.5 years). A promoter mutation showed reduced in vitro activity corresponding to lower mRNA expression in patient samples. Stimulation of the protein kinase A-pathway positively regulates the AIP promoter. Silent mutations led to abnormal splicing resulting in truncated protein or reduced AIP expression. A two-hybrid assay of protein-protein interaction of all missense variants showed variable disruption of AIP-phosphodiesterase-4A5 binding. In summary, exonic, promoter, splice-site, and large deletion mutations in AIP are implicated in 31% of families in our FIPA cohort. Functional characterization of AIP changes is important to identify the functional impact of gene sequence variants
The role of endogenous aryl hydrocarbon receptor signaling in cardiovascular physiology
The aryl hydrocarbon receptor (AHR) is an orphan nuclear receptor with a primary function of mediating xenobiotic metabolism through transcriptional activation of Phase I and Phase II drug-metabolizing enzymes. Although no high-affinity physiological activators of AHR have been discovered, the endogenous signaling of the AHR pathway is believed to play an important role in the development and function of the cardiovascular system, based on the observations on ahr gene-deficient mice. The AHR knockout mice develop cardiac hypertrophy, abnormal vascular structure in multiple organs and altered blood pressure depending on their host environment. In this review, the endogenous role of AHR in cardiovascular physiology, including heart function, vascular development and blood pressure regulation has been summarized and discussed
Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout
Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Toxicology and Applied Pharmacology 254 (2011): 1-7, doi:10.1016/j.taap.2011.04.015.This
study
assessed
the
role
of
aryl
hydrocarbon
receptor
(AHR)
affinity,
and
cytochrome
P4501A
(CYP1A)
protein
and
activity
in
polyaromatic
hydrocarbon
(PAH)-‐induced
oxidative
stress.
In
the
1-‐100
nM
concentration
range
benzo[a]pyrene
(BaP)
but
not
benzo[e]pyrene
(BeP)
competitively
displaced
2
nM
[3H]2,
3,
7,
8-‐tetrachloro-‐dibenzo-‐p-‐dioxin
from
rainbow
trout
AHR2α.
Based
on
appearance
of
fluorescent
aromatic
compounds
in
bile
over
3,
7,
14,
28
or
50
days
of
feeding
3
μg
of
BaP
or
BeP/g
fish/day,
rainbow
trout
liver
readily
excreted
these
polyaromatic
hydrocarbons
(PAHs)
and
their
metabolites
at
near
steady
state
rates.
CYP1A
proteins
catalyzed
more
than
98%
of
ethoxyresorufin-‐O-‐deethylase
(EROD)
activity
in
rainbow
trout
hepatic
microsomes.
EROD
activity
of
hepatic
microsomes
initially
increased
and
then
decreased
to
control
activities
after
50
days
of
feeding
both
PAHs.
Immunohistochemistry
of
liver
confirmed
CYP1A
protein
increased
in
fish
fed
both
PAHs
after
3
days
and
remained
elevated
for
up
to
28
days.
Neither
BaP
nor
BeP
increased
hepatic
DNA
adduct
concentrations
at
any
time
up
to
50
days
of
feeding
these
PAHs.
Comet
assays
of
blood
cells
demonstrated
marked
DNA
damage
after
14
days
of
feeding
both
PAHs
that
was
not
significant
after
50
days.
There
was
a
strong
positive
correlation
between
hepatic
EROD
activity
and
DNA
damage
in
blood
cells
over
time
for
both
PAHs.
Neither
CYP1A
protein
nor
3-‐
nitrotyrosine
(a
biomarker
for
oxidative
stress)
immunostaining
in
trunk
kidney
were
significantly
altered
by
BaP
or
BeP
after
3,
7,
14,
or
28
days.
There
was
no
clear
association
between
AHR2α
affinity
and
BaP
and
BeP-‐induced
oxidative
stress.The
Oregon
Agricultural
Experiment
Station,
Northwest
Fisheries
Science
Center,
and
RO1ES006272
from
the
National
Institute
of
Health
supported
this
work
Expression Profiling of CYP1B1 in Oral Squamous Cell Carcinoma: Counterintuitive Downregulation in Tumors
Oral Squamous Cell Carcinoma (OSCC) has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy. CYP1B1, a member of cytochrome P450 family, has been implicated in chemical carcinogenesis. There exists a general accordance that this protein is overexpressed in a variety of cancers, making it an ideal candidate for a prodrug therapy. The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits. This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This report therefore clearly demonstrates the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers and also suggest the possibility of a prophylactic therapy for oral cancer
A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests
<p>Abstract</p> <p>Background</p> <p>Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power.</p> <p>Results</p> <p>We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization.</p> <p>Conclusions</p> <p>A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.</p
Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice
We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology
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