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 tissue- specific expression of enzymes (retinaldehyde dehydrogenases, to be specific) Affects vertebrate physiological processes Development Development Cell growth and differentiation Cell growth and differentiation Morphogenesis Morphogenesis Adult tissue function Adult tissue function

Retinoid Isomer Structures All-trans Retinoic acid 9-cis Retinoic acid

How Retinoic Acid Effects Gene Expression Gene product Retinoid binding protein 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 ( E 2 stimulates cell proliferation in OVX rats) 3. Inject 3 H Thy 4. Remove uterus 24 hrs post E 2 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 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. 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 Dose of Retinoid (  M) % Failed Hatching

Embryonic Morphology on Experimental Day 6 Control ATRA 9-cis RA 10nM 100nM 1  M

Cardiac Morphology Is Dramatically Altered by 100nM ATRA Treatment 100nM ATRA Control

Cardiac Morphology Is Altered by 50nM ATRA Treatment 50nM ATRA Control

Range of Cardiac Malformations

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 All- trans and 9-cis Retinoic Acid on Medaka (Oryzias latipes) Development” BIOS 76(1), Munroe Sisters Tri-Beta Research Foundation NSF MRI