The Secret Life of The Secret Life of Vitamin AVitamin A

Holly Boettger-Tong, Wesleyan College Holly Boettger-Tong, Wesleyan College Department of BiologyDepartment of Biology

What is Vitamin A ?What is Vitamin A ?Required for normal visionRequired for normal visionreproduction,differentiation, reproduction,differentiation, homeostasishomeostasisDeficiency leads to blindness, Deficiency leads to blindness, sterilitysterilityRetinol, Retinal, Retinoic acid Retinol, Retinal, Retinoic acid all have biological activity. all have biological activity. Nature of function depends Nature of function depends upon form, converting enzymes, upon form, converting enzymes, expression of receptors for each expression of receptors for each retinoidretinoid

Retinoic AcidRetinoic AcidMetabolite of Vitamin AMetabolite of Vitamin A

Organic compound that is produced endogenously and whose Organic compound that is produced endogenously and whose production is dependent upon tissue-specific expression of production is dependent upon tissue-specific expression of enzymes (retinaldehyde dehydrogenases, to be specific) enzymes (retinaldehyde dehydrogenases, to be specific)

Affects vertebrate physiological processes Affects vertebrate physiological processes DevelopmentDevelopment Cell growth and differentiationCell growth and differentiation MorphogenesisMorphogenesis Adult tissue functionAdult tissue function

Retinoid Isomer StructuresRetinoid Isomer Structures

All-trans Retinoic acid

9-cis Retinoic acid

How Retinoic Acid Effects How Retinoic Acid Effects Gene ExpressionGene Expression

Gene product

Retinoid binding protein

Retinoic AcidReceptor – bindsligand and DNA, Influencing gene expression

What is the effect of Vitamin A What is the effect of Vitamin A on Cell Proliferation in the on Cell Proliferation in the

Uterus?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 All-trans Retinoic Acid Inhibits Estrogen Induced Uterine Myometrial and Induced Uterine Myometrial and

Stromal Cell ProliferationStromal Cell Proliferation

1. Treat animalswith 30mg/kg ATRAprior to estradiol ( E2

stimulates cellproliferation in OVX rats)3. Inject 3H Thy4. Remove uterus 24 hrspost E2 injection5. Section uterus6. Expose to emulsion7. Develop slides

Why Switch to Cell Culture Why Switch to Cell Culture Experiments?Experiments?

Advantages:Easier to work with than whole animalsMore manipulable, faster resultsGreater range of treatment concentrationsCheaperCan extend studies to humans (cultured

human cells)

Disadvantages:May not reiterate effect seen in vivoNon-physiologicalPrimary cells have limited lifespan

Effect of Retinoic Acid on Human Effect of Retinoic Acid on Human Myometrial Cell ProliferationMyometrial Cell Proliferation

Retinoic Acid Alters Uterine Retinoic Acid Alters Uterine Smooth Muscle Cell MorphologySmooth Muscle Cell Morphology

Control, Day 6 10-9 M ATRA, Day 6

Effects on Proliferation and Effects on Proliferation and Morphology are ReversibleMorphology are Reversible

A = ControlB= Day 6, RA treatedC= Day 12, ControlD= Day 12, RA discontinued

Do Retinoids Influence the Growth Do Retinoids Influence the Growth of of ImmortalizedImmortalized Human Uterine Human Uterine

Cells?Cells?

Come to Erin’s talk when she Come to Erin’s talk when she defends her Honor’s thesis. defends her Honor’s thesis.

Retinoids and DevelopmentRetinoids and DevelopmentSpecification of primary antero-Specification of primary antero-posterior axis, forming nervous system, posterior axis, forming nervous system, establishing limbsestablishing limbsControl of gene expression – produced Control of gene expression – produced at specific developmental times to yield at specific developmental times to yield a gradient of retinoic acida gradient of retinoic acidDefects in retinoic acid production or in Defects in retinoic acid production or in receptor expression result in defective receptor expression result in defective development. Excess retinoic acid is development. Excess retinoic acid is teratogenic, causing malformations teratogenic, causing malformations which are informative as to retinoid which are informative as to retinoid action in specific tissues. action in specific tissues.

The Japanese KillifishThe Japanese KillifishOryzias latipesOryzias latipes are native to Japan, Taiwan, and are native to Japan, Taiwan, and southeastern Asiasoutheastern Asia

Freshwater and common to rice paddiesFreshwater and common to rice paddies

Rapid development (ranges from 1-3 weeks) in Rapid development (ranges from 1-3 weeks) in a translucent egg makes them ideal for a translucent egg makes them ideal for determining potential teratologic effects of determining potential teratologic effects of chemicals of interestchemicals of interest

Good for students due to ease of cultureGood for students due to ease of culture

What effects occur when What effects occur when Medaka embryos in the Medaka embryos in the neurula stage of neurula stage of development are treated development are treated with all-trans and 9-cis with all-trans and 9-cis retinoic acid?retinoic acid?

Experimental DesignExperimental Design

Modified FETAX – treat embryos for three Modified FETAX – treat embryos for three days with different concentrations of days with different concentrations of retinoid (or vehicle control), then retinoid (or vehicle control), then discontinue use and observe effects of discontinue use and observe effects of treatmenttreatmentMonitor embryos daily, observing Monitor embryos daily, observing morphologymorphologyDetermine effects on hatchingDetermine effects on hatching

1 6

3 3 3 3

8 3

1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

0 0 . 0 0 1 0 . 0 1 0 . 1 1

Dose Dependent Inhibition of Dose Dependent Inhibition of Medaka Hatching by ATRA and 9-Medaka Hatching by ATRA and 9-

cis RAcis RA

Dose of Retinoid (M)

% F

aile

d H

atch

ing

Embryonic Morphology on Embryonic Morphology on Experimental Day 6Experimental Day 6

Control

ATRA

9-cis RA

10nM 100nM 1M

Cardiac MorphologyCardiac MorphologyIs Dramatically Altered by 100nM ATRA Is Dramatically Altered by 100nM ATRA

TreatmentTreatment

100nM ATRA100nM ATRAControlControl

Cardiac MorphologyCardiac MorphologyIs Altered by 50nM ATRA TreatmentIs Altered by 50nM ATRA Treatment

50nM ATRA50nM ATRA ControlControl

Range of Cardiac MalformationsRange 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 What are the Genetic Consequences of Treating Neurula Embryos with Treating Neurula Embryos with

Retinoic Acid?Retinoic Acid?

To study global changes in gene expression To study global changes in gene expression patterns we use DNA microarrayspatterns we use DNA microarraysLarge numbers of genes (from 5-10K) Large numbers of genes (from 5-10K) represented on small coated glass slides represented on small coated glass slides (chips) (chips) Assess changes in gene expression patterns in Assess changes in gene expression patterns in normal vs treated embryos (normal vs treated embryos (work in progresswork in progress))

What Secrets Have These Experiments What Secrets Have These Experiments Revealed About Retinoids?Revealed About Retinoids?

Retinoic acid inhibits smooth muscle cell Retinoic acid inhibits smooth muscle cell proliferation in both normal and benign tumor cells proliferation in both normal and benign tumor cells (implications for pharmacologic treatment of (implications for pharmacologic treatment of leiomyomas?)leiomyomas?)Deducing mechanism of action may reveal more Deducing mechanism of action may reveal more information about growth control mechanisms in information about growth control mechanisms in these cells (implications for deciphering events these cells (implications for deciphering events during pregnancy which lead to myometrial cell during pregnancy which lead to myometrial cell hyperplasia)hyperplasia)Role of retinoids in heart development poorly Role of retinoids in heart development poorly understoodunderstoodImplications for cardiac malformations in higher Implications for cardiac malformations in higher vertebrates (including man); opportunity to dissect vertebrates (including man); opportunity to dissect gene pathway leading to chambering gene pathway leading to chambering

AcknowledgementsAcknowledgements

Namita Mohanty (GH 2003, “Comparative Effects Namita Mohanty (GH 2003, “Comparative Effects of All-trans and 9-cis Retinoic Acid on Medaka of All-trans and 9-cis Retinoic Acid on Medaka ((Oryzias latipesOryzias latipes) Development” BIOS 76(1), 2005. ) Development” BIOS 76(1), 2005.

Munroe SistersMunroe Sisters

Tri-Beta Research FoundationTri-Beta Research Foundation

NSF MRI 0116189NSF MRI 0116189