All-trans retinoic acid stimulates growth of adult human keratinocytes cultured in growth factor-deficient medium, inhibits production of thrombospondin and fibronectin, and reduces adhesion.
Human epidermal keratinocytes were established in culture using a low-Ca2+ (0.15 mM), serum-free keratinocyte growth medium (KGM) as the culture medium. Early passage keratinocytes (i.e., between passages 3-8) were incubated for 1 or 2 d in KGM, in KGM supplemented with 1.4 mM Ca2+, or in growth factor-deprived keratinocyte basal medium (KBM). The cells were concomitantly treated with all-trans retinoic acid (0.1-2.5 micrograms/ml), and cell growth was quantitated at the end of the incubation period. The keratinocytes were simultaneously examined for adhesiveness and production of two extracellular matrix molecules, e.g., thrombospondin (TSP) and fibronectin (FN). Treatment with all-trans retinoic acid inhibited proliferation of keratinocytes that were rapidly growing in KGM. Proliferation was also inhibited in KGM supplemented with 1.4 mM Ca2+, but all-trans retinoic acid did not reverse the morphologic features associated with differentiation induced by high Ca2+. In contrast to these effects, all-trans retinoic acid treatment of keratinocytes in KBM, in which the cells were normally quiescent, stimulated growth. In the presence of optimal concentrations of all-trans retinoic acid (0.5 microgram/ml), the rate of keratinocyte proliferation in KBM was approximately 35% of the rate obtained in KGM (maximal proliferation rate). Keratinocyte adhesion (resistance to trypsin-mediated release from the substrate and attachment to the substrate) was inhibited by all-trans retinoic acid under all three conditions. In regard to extracellular matrix production, TSP production was inhibited by greater than 90% under all three conditions in the presence of all-trans retinoic acid. FN production was also inhibited but to a lesser degree. Concentrations of all-trans retinoic acid required to maximally inhibit keratinocyte adhesion and matrix production were higher (1.0-2.5 microgram/ml) than the concentration required to stimulate proliferation in KBM. These in vitro observations may have implications in the effects of retinoids on intact skin, including enhanced keratinocyte proliferation and thickening of the epidermis after topical application to photoaged skin and inhibition of proliferation and cell-cell cohesion after systemic administration in cases of psoriasis.
Varani
J
J
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109-0602.
Nickoloff
B J
BJ
Dixit
V M
VM
Mitra
R S
RS
Voorhees
J J
JJ
D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D004797:Enzyme-Linked Immunosorbent Assay; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects; D005353:Fibronectins / Q000096:biosynthesis*; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007633:Keratins; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D019699:Thrombospondins; D014212:Tretinoin / Q000494:pharmacology*
D003470:Culture Media; D005353:Fibronectins; D006133:Growth Substances; D008562:Membrane Glycoproteins; D019699:Thrombospondins; D014212:Tretinoin; D007633:Keratins; D002118:Calcium