The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment.
Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.
De Luca
M
M
Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
Pellegrini
G
G
Bondanza
S
S
Cremona
O
O
Savoia
P
P
Cancedda
R
R
Marchisio
P C
PC
D002118:Calcium / Q000494:pharmacology; D002455:Cell Division; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology; D005455:Fluorescent Antibody Technique; D006801:Humans; D016023:Integrins / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D009994:Osmolar Concentration
D016023:Integrins; D002118:Calcium