Transcript Retinoic Acid - Wesleyan College Faculty

The Secret Life of
Vitamin A
Holly Boettger-Tong, Wesleyan College
Department of Biology
What is Vitamin A ?
Required for normal vision
reproduction,differentiation,
homeostasis
Deficiency leads to blindness,
sterility
Retinol, Retinal, Retinoic acid
all have biological activity.
Nature of function depends
upon form, converting
enzymes, expression of
receptors for each retinoid
Retinoic Acid
Metabolite of Vitamin A
Organic compound that is produced endogenously
and whose production is dependent upon tissuespecific expression of enzymes (retinaldehyde
dehydrogenases, to be specific)
Affects vertebrate physiological processes
Development
Cell growth and differentiation
Morphogenesis
Adult tissue function
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Retinoid Isomer Structures
All-trans Retinoic acid
9-cis Retinoic acid
How Retinoic Acid Effects
Gene Expression
Retinoid binding
protein
Gene product
Retinoic Acid
Receptor – binds
ligand and DNA,
Influencing gene
expression
What is the effect of Vitamin A
on Cell Proliferation in the
Uterus?
1.Estrogen is a potent regulator of uterine
growth
2.In immature rats, all uterine cell types
proliferate in response to estrogen treatment
3.What effect, if any, does retinoic acid have on
estrogen-induced uterine cell proliferation?
All-trans Retinoic Acid Inhibits Estrogen
Induced Uterine Myometrial and
Stromal Cell Proliferation
1. Treat animals
with 30mg/kg ATRA
prior to estradiol ( E2
stimulates cell
proliferation in OVX rats)
3. Inject 3H Thy
4. Remove uterus 24 hrs
post E2 injection
5. Section uterus
6. Expose to emulsion
7. Develop slides
Why Switch to Cell Culture
Experiments?
Advantages:
Easier to work with than whole animals
More manipulable, faster results
Greater range of treatment concentrations
Cheaper
Can extend studies to humans (cultured
human cells)
Disadvantages:
May not reiterate effect seen in vivo
Non-physiological
Primary cells have limited lifespan
Effect of Retinoic Acid on Human
Myometrial Cell Proliferation
Retinoic Acid Alters Uterine
Smooth Muscle Cell Morphology
Control, Day 6
10-9 M ATRA, Day 6
Effects on Proliferation and
Morphology are Reversible
A = Control
B= Day 6, RA treated
C= Day 12, Control
D= Day 12, RA discontinued
Do Retinoids Influence the Growth
of Immortalized Human Uterine
Cells?
Come to Erin’s talk when she
defends her Honor’s thesis.
Retinoids and Development
Specification of primary antero-posterior
axis, forming nervous system, establishing
limbs
Control of gene expression – produced at
specific developmental times to yield a
gradient of retinoic acid
Defects in retinoic acid production or in
receptor expression result in defective
development. Excess retinoic acid is
teratogenic, causing malformations which
are informative as to retinoid action in
specific tissues.
The Japanese Killifish
Oryzias latipes are native to Japan, Taiwan, and
southeastern Asia
Freshwater and common to rice paddies
Rapid development (ranges from 1-3 weeks) in
a translucent egg makes them ideal for
determining potential teratologic effects of
chemicals of interest
Good for students due to ease of culture
What effects occur when
Medaka embryos in the
neurula stage of development
are treated with all-trans and
9-cis retinoic acid?
Experimental Design
Modified FETAX – treat embryos for three
days with different concentrations of
retinoid (or vehicle control), then
discontinue use and observe effects of
treatment
Monitor embryos daily, observing
morphology
Determine effects on hatching
Dose Dependent Inhibition of
Medaka Hatching by ATRA and 9-cis RA
100
% Failed Hatching
100
83
80
60
40
33
20
33
16
0
0
0.001
0.01
0.1
Dose of Retinoid (mM)
1
Embryonic Morphology on Experimental Day 6
Control
ATRA
9-cis RA
10nM
100nM
1mM
Cardiac Morphology
Is Dramatically Altered by 100nM ATRA
Treatment
Control
100nM ATRA
Cardiac Morphology
Is Altered by 50nM ATRA Treatment
50nM ATRA
Control
Range of Cardiac Malformations
Normally
Chambered
Hearts
Heart
Looping
Heart Strings/
Cardiac Tissue
Masses
Control
+++
-
-
10nM ATRA/9-cis
+++
-
-
50nM ATRA/9-cis
++
+++
-
100nM ATRA/9-cis
-
++
+++
1µM ATRA/9-cis
-
-
-
What are the Genetic Consequences of
Treating Neurula Embryos with Retinoic
Acid?
To study global changes in gene expression
patterns we use DNA microarrays
Large numbers of genes (from 5-10K)
represented on small coated glass slides
(chips)
Assess changes in gene expression patterns in
normal vs treated embryos (work in progress)
What Secrets Have These Experiments
Revealed About Retinoids?
Retinoic acid inhibits smooth muscle cell
proliferation in both normal and benign tumor cells
(implications for pharmacologic treatment of
leiomyomas?)
Deducing mechanism of action may reveal more
information about growth control mechanisms in
these cells (implications for deciphering events
during pregnancy which lead to myometrial cell
hyperplasia)
Role of retinoids in heart development poorly
understood
Implications for cardiac malformations in higher
vertebrates (including man); opportunity to dissect
gene pathway leading to chambering
Acknowledgements
Namita Mohanty (GH 2003, “Comparative Effects of Alltrans and 9-cis Retinoic Acid on Medaka (Oryzias
latipes) Development” BIOS 76(1), 2005.
Munroe Sisters
Tri-Beta Research Foundation
NSF MRI 0116189