Serum-free cultured keratinocytes fail to organize fibronectin matrix and possess different distribution of beta-1 integrins.
The development of serum free medium formulation for culturing keratinocytes was a breakthrough in achieving a high number of epidermal cells for experimental and therapeutic studies, in particular to support the wound healing process. It is not clear, however, if switching the cells to highly proliferative phenotype may reflect change in other cellular functions important for the wound repair as their adhesive interactions with the extracellular matrix components. Remodelling of the extracellular matrix, particularly of fibronectin plays an essential role for guiding the cells during wound healing. The molecular mechanisms for organization of this provisional fibronectin matrix, however, are still not clear. We found that keratinocytes in serum containing medium, although in fewer numbers than fibroblasts, were able to remove adsorbed fluorescent labelled fibronectin from the substratum and reorganize it in a fibrilar pattern along the cell periphery. After 3 days the secreted fibronectin had also been organized as matrix-like fibers and as clusters deposited on the substratum after migrating cells. In contrast, serum free cultured keratinocytes fail to organize pre-adsorbed fluorescent labelled fibronectin, as well as the secreted fibronectin, although they grow very well under these conditions. Switching the cells to serum containing medium initiates the removal of fluorescent labelled fibronectin from the substratum, however without reorganization in fibrillar pattern. Most likely, these keratinocytes remove fluorescent labelled fibronectin by the expression of proteolytic activity, rather than with the mechanical function of beta(1) integrins. The latter were diffusely dispersed in serum containing conditions and tend to organize in focal adhesions in serum free cultured cells. We assumed their transient expression and different affinity state might be important for the keratinocyte migration and matrix assembly mechanism.
Altankov
G
G
Institute of Biophysics, Bulgarian Academy of Sciences, Acad. G. Bonchev Bl. 21, BG-1113 Sofia, Bulgaria. altankov@obzor.bio21.bas.bg
Hecht
J
J
Dimoudis
N
N
D001790:Blood Physiological Phenomena; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion; D048429:Cell Size; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000378:metabolism*; D005312:Fetal Blood / Q000502:physiology; D005340:Fibrinogen / Q000032:analysis; D005353:Fibronectins / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D019012:Integrin beta1 / Q000032:analysis*; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008856:Microscopy, Fluorescence; D016596:Vinculin / Q000032:analysis
D003470:Culture Media; D016895:Culture Media, Serum-Free; D005353:Fibronectins; D005456:Fluorescent Dyes; D019012:Integrin beta1; D016596:Vinculin; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium