The effect of agent-induced differentiation on cellular retinoic acid-binding protein in primary rat keratinocyte cultures.

Abstract

Retinoids are vitamin A compounds which demonstrate anti-tumor promoting capacity and the ability to direct differentiation in epithelial cells. The molecular mechanism of this action remains unknown, but may be related to intracellular binding proteins. Primary rat keratinocyte cultures have been utilized to investigate the effect of retinoic acid on epidermal differentiation. Various calcium levels were used to regulate proliferation and differentiation. Retinoic acid added to the culture medium directs differentiation away from the stratification and keratinization of terminally differentiating cultures, while the addition of the tumor promoter TPA (phorbol actetate) will accelerate the process. A pretreatment with retinoic acid will neutralize the effect of TPA. The cultures also contain the binding protein for retinoic acid, CRABP. This dissertation provides data to further characterize the influence of retinoic acid on selected biochemical parameters and its ability to antagonize TPA effects in primary rat keratinocyte cultures. It also reports the modulation of CRABP by agents which direct keratinocyte differentiation. Retinoic acid will enter the keratinocyte, distribute throughout the cell and direct new protein synthesis. When administered prior to TPA, it will allow the cell to maintain protein synthesis and prevent both the loss of cells from the culture and the atypical morphology associated with exposure to the tumor promoter. Treatment of cells with either retinoic acid or TPA also alters the binding characteristics of CRABP as assayed on a sucrose gradient. CRABP was detected by ultracentrifugation as a peak of ($\sp3$H) -retinoic acid in the 2S region of a 5 to 20% sucrose gradient. Culture conditions favoring differentiation along the retinoid or keratinizing pathways increase the amount of specifically bound radioactivity in the 2S peak and have been interpreted as an increase in CRABP activity. TPA decreases the amount of ($\sp3$H) -retinoic acid binding. This suggests that CRABP may be influenced by cellular differentiation and by agents which affect this process. A dextran-charcoal assay was employed to further characterize the apparent change in binding capacity. Results indicate that retinoic acid and high calcium increase the number of binding sites while the alterations associated with TPA may be related to a change in binding characteristics. Limitations associated with this keratinocyte system, also discussed, preclude further characterization of this effect.