The long term objective of the proposed studies is to understand the underlying mechanisms controlling epidermal cell function. The hypothesis is that the fatty acid composition of basal cell membrane phospholipids controls membrane fluidity. Altered membrane fluidity would alter the dynamics of membrane protein receptor activity, resulting in abnormal cell function. Biological membranes consist of a lipid bilayer composed of phospholipids with fatty acids in the 1 and 2 carbon position. Linoleic acid (18:2, n-6) and arachidonic acid (20:4) are essential, and are derived from the diet. Many membrane bound proteins are enzymes and proteins involved in signal transduction and their function is known to be controlled by cell membrane fluidity (viscosity). Benign skin diseases are characterized by hyperproliferation and abnormal differentiation, postulated to result from abnormal signal transduction mechanisms. One such disease, psoriasis, has an abnormal fatty acid membrane profile, and is characterized by second messenger and signaling abnormalities. An in vitro technic using no serum results in the extremely rapid growth of keratinocytes which do not differentiate. These cell are extremely EFA deficient. Using this in vitro system studies are proposed to define the effect of cell membrane fluidity on human keratinocytes which do not differentiate. These cell are extremely EFA deficient. Using this in vitro system studies are proposed to define the effect of cell membrane fluidity on human keratinocyte growth and second messenger signaling activities.
The aims are first, to determine if marked changes occur in other key membrane lipids of these cells (sterols, sphingolipids). Second, protocols for restoring in vivo ratio of the fatty acids to the cells will be developed. Third, quantitation of the fluidity of the membranes of normal, EFA deficient, and partially fatty acid restored cells will be done using Electron Spin Resonance Spectroscopy and a number of spin labeled probes. Fourth, second messenger systems and signaling mechanisms of EFA deficient and restored cells will be studied. These are: expression of EGF receptor and associated tyrosine kinase activity; cAMP and cGMP systems; phospholipase A2 and C systems, all of which are abnormally expressed in psoriasis. The technologies of in vitro cell growth, HPLC analysis, TLC, ESR spectroscopy, PAGE, receptor affinity determinations and enzyme assays will be used. Recent studies using ESR spectroscopy demonstrate that altered cell membrane fatty acid content does alter the viscosity of the keratinocyte membrane. Medium changes designed to alter cell fatty acid profiles were successful in altering the fatty acid content of the cultures, changes accompanied by demonstrated changes in membrane fluidity. Thus new evidence supports the hypothesis that basal cell fatty acid composition controls membrane fluidity. The studies described in this application propose to study the mechanisms of this control and its relationship to epidermal cell function.
|Hee Chung, S; Terashi, H; Rhodes, L M et al. (2001) The effect of three Korean traditional medicines on the growth rate of cultured human keratinocytes. J Ethnopharmacol 74:53-61|
|Marcelo, C L; Dunham, W R (1997) Retinoic acid stimulates essential fatty acid-supplemented human keratinocytes in culture. J Invest Dermatol 108:758-62|
|Fulbright, R M; Axelrod, D; Dunham, W R et al. (1997) Fatty acid alteration and the lateral diffusion of lipids in the plasma membrane of keratinocytes. Exp Cell Res 233:128-34|
|Dunham, W R; Klein, S B; Rhodes, L M et al. (1996) Oleic acid and linoleic acid are the major determinants of changes in keratinocyte plasma membrane viscosity. J Invest Dermatol 107:332-5|
|Garner, W L; Oyatsu, Y; Zuccaro, C et al. (1995) The effect of essential fatty acid supplementation on keratinocyte replication. Prostaglandins Leukot Essent Fatty Acids 52:349-55|
|Marcelo, C L; Rhodes, L M; Dunham, W R (1994) Normalization of essential-fatty-acid-deficient keratinocytes requires palmitic acid. J Invest Dermatol 103:564-8|
|Marcelo, C L; Dunham, W R (1993) Fatty acid metabolism studies of human epidermal cell cultures. J Lipid Res 34:2077-90|
|Marcelo, C L; Duell, E A; Rhodes, L M et al. (1992) In vitro model of essential fatty acid deficiency. J Invest Dermatol 99:703-8|
|Hadley, G A; Marcelo, C L; Steinmuller, D (1988) Monoclonal antibodies to tissue-restricted autoantigens in the mouse epidermis. Hybridoma 7:441-51|